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Abstract:

Oligomeric compounds, compositions and methods are provided for
modulating the expression of eIF4E. The antisense compounds may be
single- or double-stranded and are targeted to nucleic acid encoding
eIF4E. Methods of using these compounds for modulation of eIF4E
expression and for diagnosis and treatment of diseases and conditions
associated with expression of eIF4E are provided.

Claims:

1. An antisense oligonucleotide consisting of 12 to 30 linked nucleosides
and having a nucleobase sequence at least 95% complementary within the 3'
untranslated region (3'UTR) of a nucleic acid encoding human eIF4E,
wherein the antisense oligonucleotide comprises at least one chemically
modified sugar moiety, internucleoside linkage, or nucleobase.

13. The antisense oligonucleotide of claim 1, wherein the antisense
oligonucleotide is in the form of a pharmaceutically acceptable salt.

14. A method for treating a condition or disease associated with eIF4E
expression or overexpression with the antisense oligonucleotide of claim
1, comprising administering the antisense oligonucleotide to a subject in
need thereof.

15. The method of claim 14, wherein said condition or disease associated
with eIF4E expression or overexpression is a hyperproliferative condition
or disease.

16. The method of claim 15, wherein said hyperproliferative condition or
disease is selected from the group consisting of breast cancer, head and
neck cancer, colorectal cancer, prostate cancer, lung cancer, bladder
cancer, ovarian cancer, renal cancer, and glioblastoma.

17. A method for treating a condition or disease associated with eIF4E
expression or overexpression with the antisense oligonucleotide of claim
11, comprising administering the antisense oligonucleotide to a subject
in need thereof.

18. The method of claim 17, wherein said hyperproliferative condition or
disease is selected from the group consisting of breast cancer, head and
neck cancer, colorectal cancer, prostate cancer, lung cancer, bladder
cancer, ovarian cancer, renal cancer, and glioblastoma.

19. A method for treating a condition or disease associated with eIF4E
expression or overexpression with the antisense oligonucleotide of claim
12, comprising administering the antisense oligonucleotide to a subject
in need thereof.

20. The method of claim 19, wherein said hyperproliferative condition or
disease is selected from the group consisting of breast cancer, head and
neck cancer, colorectal cancer, prostate cancer, lung cancer, bladder
cancer, ovarian cancer, renal cancer, and glioblastoma.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.
13/548,784, filed Jul. 13, 2012, which is a continuation of U.S.
application Ser. No. 12/550,479, filed Aug. 31, 2009, which is a
continuation of U.S. application Ser. No. 12/184,379, filed Aug. 1, 2008,
now U.S. Pat. No. 7,601,700, which is a divisional of U.S. application
Ser. No. 10/571,339, filed Nov. 29, 2006, now U.S. Pat. No. 7,425,554,
which is a United States National Phase filing under 35 U.S.C. 371
claiming priority to International Application No. PCT/US2004/030436,
filed Sep. 17, 2004, and claims the benefit of priority under 35 U.S.C.
119(e) to U.S. provisional patent application Ser. No. 60/576,534, filed
Jun. 3, 2004 and U.S. provisional patent application Ser. No. 60/504,110,
filed Sep. 18, 2003. Each of the above applications is incorporated
herein by reference in its entirety.

SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a file
entitled RTS0552USC3SEQ.txt, created on Feb. 28, 2013 which is 92 Kb in
size. The information in the electronic format of the sequence listing is
incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention provides compositions and methods for
modulating the expression of eIF4E. In particular, this invention relates
to single- or double-stranded antisense compounds, particularly
oligonucleotide compounds, which hybridize with nucleic acid molecules
encoding eIF4E. Such compounds are shown herein to modulate the
expression of eIF4E.

BACKGROUND OF THE INVENTION

[0004] Eukaryotic gene expression must be regulated such that cells can
rapidly respond to a wide range of different conditions. The process of
mRNA translation is one step at which gene expression is highly
regulated. In response to hormones, growth factors, cytokines and
nutrients, animal cells generally activate translation in preparation for
the proliferative response. The rate of protein synthesis typically
decreases under stressful conditions, such as oxidative or osmotic
stress, DNA damage or nutrient withdrawal. Activation or suppression of
mRNA translation occurs within minutes and control over this process is
thought to be exerted at the initiation phase of protein synthesis
(Rosenwald et al., Oncogene, 1999, 18, 2507-2517; Strudwick and Borden,
Differentiation, 2002, 70, 10-22).

[0005] Translation initiation necessitates the coordinated activities of
several eukaryotic initiation factors (eIFs), proteins which are
classically defined by their cytoplasmic location and ability to regulate
the initiation phase of protein synthesis. One of these factors,
eukaryotic initiation factor 4E (eIF4E) (also known as eukaryotic
translation initiation factor 4E, eukaryotic translation initiation
factor 4E-like 1 (eIF4EL1), cap-binding protein (CBP) and messenger RNA
cap-binding protein) was initially isolated as a 25 kDa mRNA cap-binding
protein involved in translation (Rychlik et al., Proc. Natl. Acad. Sci.
USA, 1987, 84, 945-949) and has since become one of the most
highly-characterized eIFs. eIF4E, present in limiting amounts relative to
other initiation factors, is one component of the eIF4F initiation
complex, which is also comprised of a scaffold protein eIF4G and the RNA
helicase eIF4A. In the cytoplasm, eIF4E catalyzes the rate-limiting step
of cap-dependent protein synthesis by specifically binding to the 5'
terminal 7-methyl GpppX cap structure present on nearly all mature
cellular mRNAs, which serves to deliver the mRNAs to the eIF4F complex.
Once bound, the eIF4F complex scans from the 5' to the 3' end of the cap,
permitting the RNA helicase activity of eIF4A to resolve any secondary
structure present in the 5' untranslated region (UTR), thus revealing the
translation initiation codon and facilitating ribosome loading onto the
mRNA (Graff et al., Clin. Exp. Metastasis, 2003, 20, 265-273; Strudwick
et al., Differentiation, 2002, 70, 10-22).

[0007] A growing number of observations suggest that translation factors
localize and function in the nucleus, as well as in the cytoplasm.
Transcription and translation are traditionally considered to be
spatially separated in eukaryotes; however, coupled transcription and
translation is observed within the nuclei of mammalian cells (Iborra et
al., Science, 2001, 293, 1139-1142). A fraction of eIF4E localizes to the
nucleus, suggesting that this translation factor may exhibit some of its
control over translation in the nucleus (Lejbkowicz et al., Proc. Natl.
Acad. Sci. USA, 1992, 89, 9612-9616). eIF4E is imported into the nucleus
through the importin alpha/beta pathway by the nucleoplasmic shuttling
protein eIF4E-transporter (4E-T) (Dostie et al., Embo J., 2000, 19,
3142-3156). In the nucleus, eIF4E can be directly bound by the
promyelocytic leukemia protein (PML), an important regulator of mammalian
cell growth and apoptosis (Cohen et al., Embo J., 2001, 20, 4547-4559).
PML, through its RING domain, modulates eIF4E activity by greatly
reducing its affinity for the 5' cap structure of mRNAs (Cohen et al.,
Embo J., 2001, 20, 4547-4559).

[0009] eIF4E function is an essential determinant of overall cell protein
synthesis and growth (De Benedetti et al., Mol. Cell Biol., 1991, 11,
5435-5445). In normal cells, eIF4E is present in limiting amounts, which
restricts translation. mRNAs which encode proteins necessary for cell
growth and survival typically contain a complex, highly structured 5'
UTR, which renders these mRNAs poor substrates for translation. Many of
these mRNAs, however, are well translated in the presence of excess eIF4E
and are also upregulated by tumors (Graff and Zimmer, Clin. Exp.
Metastasis, 2003, 20, 265-273). The translation of mRNAs related to cell
differentiation may also be enhanced by eIF4E, as increased levels of
eIF4E are found in some differentiating cell lines, including epithelial
lung tumor cell lines (Walsh et al., Differentiation, 2003, 71, 126-134).

[0012] Inhibition of eIF4E expression and activity has been accomplished
through the use of antisense mechanisms. Antisense oligonucleotides
equipped with 3'-overhanging nucleotides modulate the binding of eIF4E to
5'-capped oligoribonucleotides (Baker et al., J. Biol. Chem., 1992, 267,
11495-11499). Introduction into HeLa cells of an episomal vector
engineered to express an oligonucleotide complementary to 20 nucleotides
in the translation start region of eIF4E reduces levels of eIF4E and
concomitantly decreases the rates of cell growth and protein synthesis,
demonstrating that eIF4E is required for cell proliferation (Bommer et
al., Cell. Mol. Biol. Res., 1994, 40, 633-641; De Benedetti et al., Mol.
Cell. Biol., 1991, 11, 5435-5445). Levels of eIF4G, the scaffold protein
component of the eIF4F complex, are also reduced. Despite the diminished
levels of translation following inhibition of eIF4E, certain proteins
continue to be synthesized, and many of these have been identified as
stress-inducible or heat-shock proteins (Joshi-Barve et al., J. Biol.
Chem., 1992, 267, 21038-21043). The same vector reduces eIF4E by 50 to 60
percent in rat embryo fibroblasts, which is sufficient to inhibit
ras-mediated transformation and tumorigenesis of these cells (Graff et
al., Int. J. Cancer, 1995, 60, 255-263; Rinker-Schaeffer et al., Int. J.
Cancer, 1993, 55, 841-847). Furthermore, ODC translation and polyamine
transport are diminished, an observation that provides a link between
ras-induced malignancy, eIF4E activity and polyamine metabolism (Graff et
al., Biochem. Biophys. Res. Commun., 1997, 240, 15-20). Stable
transformation of a mammary carcinoma line and a head and neck squamous
cell carcinoma cell line with the eIF4E antisense vector results in
reduction fibroblast growth factor-2 (FGF-2) expression and in inhibition
of tumorigenic and angiogenic capacity of the cells in mice, suggesting a
causal role for eIF4E in tumor vascularization (DeFatta et al.,
Laryngoscope, 2000, 110, 928-933; Nathan et al., Oncogene, 1997, 15,
1087-1094).

[0013] Targeted inactivation of a Caenorhabditis elegans homolog of human
eIF4E, IFE-3, with small interfering RNA injected into young adult worms
leads to embryonic lethality in 100% of the progeny (Keiper et al., J.
Biol. Chem., 2000, 275, 10590-10596). Small interfering double-stranded
RNA targeted to eIF4E has also revealed that lack of eIF4E regulation
participates in cellular transformation. Functional inactivation of eIF4E
using a gene-specific 21-nucleotide small interfering RNA targeted to a
portion of the coding region of human eIF4E results in a significant
reduction of anchorage-independent growth of malignant cholangiocytes, a
phenotype associated with transformed cells. In addition, phosphorylation
of eIF4E in malignant cholangiocytes is dependent upon p38 MAP kinase
signaling, demonstrating a link between p38 MAP kinase signaling and the
regulation of protein synthesis in the process of cholangiocarcinoma
growth (Yamagiwa et al., Hepatology, 2003, 38, 158-166).

[0014] Further evidence that inhibition of eIF4E activity reduces the
tumorigenic potential of cells is seen in breast cancer cells that
express a constitutively active form of the eIF4E inhibitor 4EBP-1, which
leads to cell cycle arrest associated with downregulation of cyclin D1
and upregulation of the cyclin-dependent kinase p27.sup.Kip1 (Jiang et
al., Cancer Cell Int., 2003, 3, 2). The overexpression of 4E-BP1 in
gastrointestinal cancers, where eIF4E levels are significantly higher
than in normal tissue, is correlated with a reduction in distant
metastases (Martin et al., Int. J. Biochem. Cell. Biol., 2000, 32,
633-642).

[0015] U.S. Pat. No. 5,646,009 claims and discloses a hybrid vector in
which one DNA segment encodes a cap-binding protein consisting of eIF4E,
eIF4E factor or a mutant thereof. This patent also discloses a nucleic
acid sequence encoding a human eIF4E.

[0016] Disclosed in U.S. Pat. No. 6,171,798 is a method for treating
cancer in a patient by administering to cancer cells an antisense
construct comprising at least 12 nucleotides of a coding sequence of a
gene selected from a group containing a human eIF4E, in 3' to 5'
orientation with respect to a promotor controlling its expression.

[0017] U.S. Pat. No. 6,596,854 claims and discloses isolated nucleic acid
molecules encoding variants of human eIF4E, wherein said variants have
amino acid substitutions in the regions of amino acids 112 and 114-121,
or position 118, or position 119, or position 115 or position 121.

[0018] European patent application 1 033 401 and Japanese patent
application 2001269182 claim a purified nucleic acid comprising at least
10 consecutive nucleotides of a sequence selected from a group of
EST-related sequences which includes a portion of a nucleic acid molecule
encoding human eIF4E. These publications also disclose the preparation
and use of antisense constructs and oligonucleotides to be used in gene
therapy.

[0019] PCT publications WO 01/96388 and WO 01/96389 disclose and claim
isolated polynucleotides comprising a sequence selected from: sequences,
complements of sequences, sequences consisting of at least 20 contiguous
residues of a sequence, sequences that hybridize to a sequence, or
sequences having at least 75% or at least 95% identity to a sequence,
provided in the sequence listing, which includes a nucleic acid molecule
encoding a human eIF4E. This publication also claims a method for the
treatment of a cancer in a patient, comprising administering to the
patient a composition of the claimed polynucleotides.

[0020] PCT publication WO 03/039443 claims and discloses a method for the
preparation of a pharmaceutical composition for the treatment of leukemia
characterized in that an antisense oligonucleotide complementary to a
polynucleotide encoding a protein corresponding to marker, selected from
a group including a human eIF4E nucleic acid molecule, is admixed with
pharmaceutical compounds.

[0022] Disclosed in U.S. pre-grant publication 20030144190 are antisense
molecules which may be used to decrease or abrogate the expression of a
nucleic acid sequence or protein of the invention, including eIF4E. Also
disclosed are a plasmid encoding eIF4E antisense mRNA and cultured rat
fibroblasts constitutively expressing this plasmid.

[0023] As a consequence of eIF4E involvement in many diseases, there
remains a long felt need for additional agents capable of effectively
regulating eIF4E. As such, inhibition is especially important in the
treatment of cancer, given that the upregulation of expression of eIF4E
is associated with so many different types of cancer.

[0024] Antisense technology is an effective means for reducing the
expression of specific gene products and has been proven to be uniquely
useful in a number of therapeutic, diagnostic, and research applications.
The present invention provides compositions and methods for modulating
eIF4E expression.

SUMMARY OF THE INVENTION

[0025] The present invention is directed to oligomeric compounds, such as
antisense compounds, and pharmaceutically acceptable salts thereof, which
are targeted to a nucleic acid molecule encoding eIF4E and which inhibit
the expression of eIF4E. The oligomeric compounds may be RNA-like or
DNA-like oligomeric compounds, including oligonucleotides. The oligomeric
compounds may be single-stranded or partially or wholly double-stranded
oligomeric compounds, and may be chemically modified or unmodified.
Pharmaceutical and other compositions comprising these compounds are also
provided.

[0026] Further provided are methods of screening for modulators of eIF4E
and methods of modulating the expression of eIF4E in cells, tissues or
animals comprising contacting said cells, tissues or animals with one or
more of the compounds or compositions of the invention. Methods of
treating an animal, particularly a human are also set forth herein. Such
methods comprise administering a therapeutically or prophylactically
effective amount of one or more of the compounds or compositions of the
invention.

DETAILED DESCRIPTION OF THE INVENTION

A. Overview of the Invention

[0027] The present invention employs oligomeric compounds, such as
antisense compounds, single- or double-stranded oligonucleotides and
similar species, for use in modulating the function or effect of nucleic
acid molecules encoding eIF4E. This is accomplished by providing
oligomeric compounds which specifically hybridize with one or more
nucleic acid molecules encoding eIF4E. As used herein, the terms "target
nucleic acid" and "nucleic acid molecule encoding eIF4E" have been used
for convenience to encompass DNA encoding eIF4E, RNA (including pre-mRNA
and mRNA or portions thereof) transcribed from such DNA, and also cDNA
derived from such RNA. This modulation of function of a target nucleic
acid by compounds that hybridize to it is generally referred to as
"antisense".

[0028] The functions of DNA to be interfered with can include replication
and transcription. Replication and transcription, for example, can be
from an endogenous cellular template, a vector, a plasmid construct or
otherwise. The functions of RNA to be interfered with can include
functions such as translocation of the RNA to a site of protein
translation, translocation of the RNA to sites within the cell which are
distant from the site of RNA synthesis, translation of protein from the
RNA, and catalytic activity or complex formation involving the RNA which
may be engaged in or facilitated by the RNA. One result of such
interference with target nucleic acid function is modulation of the
expression of eIF4E. In the context of the present invention,
"modulation" and "modulation of expression" mean either an increase
(stimulation) or a decrease (inhibition) in the amount or levels of a
nucleic acid molecule encoding the gene, e.g., DNA or RNA Inhibition is
one form of modulation of expression and mRNA is often a target nucleic
acid.

[0029] In the context of this invention, "hybridization" means the pairing
of substantially complementary strands of oligomeric compounds. In the
present invention, one mechanism of pairing involves hydrogen bonding,
which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen
bonding, between complementary nucleoside or nucleotide bases
(nucleobases) of the strands of oligomeric compounds. For example,
adenine and thymine are complementary nucleobases which pair through the
formation of hydrogen bonds. Hybridization can occur under varying
circumstances.

[0030] An antisense compound is "specifically hybridizable" when binding
of the compound to the target nucleic acid interferes with the normal
function of the target nucleic acid to cause a loss of activity, and
there is a sufficient degree of complementarity to avoid non-specific
binding of the antisense compound to non-target nucleic acid sequences
under conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or therapeutic
treatment, and under conditions in which assays are performed in the case
of in vitro assays.

[0031] In the present invention the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under which a
compound of the invention will hybridize to its target sequence, but to a
minimal number of other sequences. Stringent conditions are
sequence-dependent and will be different in different circumstances and
in the context of this invention, "stringent conditions" under which
oligomeric compounds hybridize to a target sequence are determined by the
nature and composition of the oligomeric compounds and the assays in
which they are being investigated. In general, stringent hybridization
conditions comprise low concentrations (<0.15M) of salts with
inorganic cations such as Na++ or K++(i.e., low ionic strength),
temperature higher than 20°-25° C. below the Tm of the
oligomeric compound:target sequence complex, and the presence of
denaturants such as formamide, dimethylformamide, dimethyl sulfoxide, or
the detergent sodium dodecyl sulfate (SDS). For example, the
hybridization rate decreases 1.1% for each 1% formamide. An example of a
high stringency hybidization condition is 0.1× sodium
chloride-sodium citrate buffer (SSC)/0.1% (w/v) SDS at 60° C. for
30 minutes.

[0032] "Complementary," as used herein, refers to the capacity for precise
pairing between two nucleobases on one or two oligomeric strands. For
example, if a nucleobase at a certain position of an antisense compound
is capable of hydrogen bonding with a nucleobase at a certain position of
a target nucleic acid, said target nucleic acid being a DNA, RNA, or
oligonucleotide molecule, then the position of hydrogen bonding between
the oligonucleotide and the target nucleic acid is considered to be a
complementary position. The oligomeric compound and the further DNA, RNA,
or oligonucleotide molecule are complementary to each other when a
sufficient number of complementary positions in each molecule are
occupied by nucleobases which can hydrogen bond with each other. Thus,
"specifically hybridizable" and "complementary" are terms which are used
to indicate a sufficient degree of precise pairing or complementarity
over a sufficient number of nucleobases such that stable and specific
binding occurs between the oligomeric compound and a target nucleic acid.

[0033] It is understood in the art that the sequence of an oligomeric
compound need not be 100% complementary to that of its target nucleic
acid to be specifically hybridizable. Moreover, an oligonucleotide may
hybridize over one or more segments such that intervening or adjacent
segments are not involved in the hybridization event (e.g., a loop
structure, mismatch or hairpin structure). The oligomeric compounds of
the present invention comprise at least about 70%, or at least about 75%,
or at least about 80%, or at least about 85%, or at least about 90%, or
at least about 95%, or at least about 99% sequence complementarity to a
target region within the target nucleic acid sequence to which they are
targeted. For example, an antisense compound in which 18 of 20
nucleobases of the antisense compound are complementary to a target
region, and would therefore specifically hybridize, would represent 90
percent complementarity. In this example, the remaining noncomplementary
nucleobases may be clustered or interspersed with complementary
nucleobases and need not be contiguous to each other or to complementary
nucleobases. As such, an antisense compound which is 18 nucleobases in
length having 4 (four) noncomplementary nucleobases which are flanked by
two regions of complete complementarity with the target nucleic acid
would have 77.8% overall complementarity with the target nucleic acid and
would thus fall within the scope of the present invention. Percent
complementarity of an antisense compound with a region of a target
nucleic acid can be determined routinely using BLAST programs (basic
local alignment search tools) and PowerBLAST programs known in the art
(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,
Genome Res., 1997, 7, 649-656). Percent homology, sequence identity or
complementarity, can be determined by, for example, the Gap program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, Madison Wis.), using default
settings, which uses the algorithm of Smith and Waterman (Adv. Appl.
Math., 1981, 2, 482-489).

[0034] The oligomeric compounds of the present invention also include
variants in which a different base is present at one or more of the
nucleotide positions in the compound. For example, if the first
nucleotide is an adenosine, variants may be produced which contain
thymidine, guanosine or cytidine at this position. This may be done at
any of the positions of the antisense compound. These compounds are then
tested using the methods described herein to determine their ability to
inhibit expression of eIF4E mRNA.

[0035] In some embodiments, homology, sequence identity or
complementarity, between the antisense compound and target is from about
50% to about 60%. In some embodiments, homology, sequence identity or
complementarity, is from about 60% to about 70%. In some embodiments,
homology, sequence identity or complementarity, is from about 70% to
about 80%. In some embodiments, homology, sequence identity or
complementarity, is from about 80% to about 90%. In some embodiments,
homology, sequence identity or complementarity, is about 90%, about 92%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about
100%.

B. Compounds of the Invention

[0036] In the context of the present invention, the term "oligomeric
compound" refers to a polymeric structure capable of hybridizing to a
region of a nucleic acid molecule. This term includes oligonucleotides,
oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and
chimeric combinations of these. Oligomeric compounds are routinely
prepared linearly but can be joined or otherwise prepared to be circular
and may also include branching. Oligomeric compounds can include
double-stranded constructs such as, for example, two strands hybridized
to form double-stranded compounds or a single strand with sufficient self
complementarity to allow for hybridization and formation of a fully or
partially double-stranded compound. In one embodiment of the invention,
double-stranded antisense compounds encompass short interfering RNAs
(siRNAs). As used herein, the term "siRNA" is defined as a
double-stranded compound having a first and second strand and comprises a
central complementary portion between said first and second strands and
terminal portions that are optionally complementary between said first
and second strands or with the target mRNA. Each strand may be from about
8 to about 80 nucleobases in length, 10 to 50 nucleobases in length, 12
or 13 to 30 nucleobases in length, 12 or 13 to 24 nucleobases in length
or 19 to 23 nucleobases in length. The central complementary portion may
be from about 8 to about 80 nucleobases in length, 10 to 50 nucleobases
in length, 12 or 13 to 30 nucleobases in length, 12 or 13 to 24
nucleobases in length or 19 to 23 nucleobases in length. The terminal
portions can be from 1 to 6 nucleobases in length. The siRNAs may also
have no terminal portions. The two strands of an siRNA can be linked
internally leaving free 3' or 5' termini or can be linked to form a
continuous hairpin structure or loop. The hairpin structure may contain
an overhang on either the 5' or 3' terminus producing an extension of
single-stranded character.

[0037] In one embodiment of the invention, double-stranded antisense
compounds are canonical siRNAs. As used herein, the term "canonical
siRNA" is defined as a double-stranded oligomeric compound having a first
strand and a second strand each strand being 21 nucleobases in length
with the strands being complementary over 19 nucleobases and having on
each 3' termini of each strand a deoxy thymidine dimer (dTdT) which in
the double-stranded compound acts as a 3' overhang.

[0038] In another embodiment, the double-stranded antisense compounds are
blunt-ended siRNAs. As used herein the term "blunt-ended siRNA" is
defined as an siRNA having no terminal overhangs. That is, at least one
end of the double-stranded compound is blunt. siRNAs whether canonical or
blunt act to elicit dsRNAse enzymes and trigger the recruitment or
activation of the RNAi antisense mechanism. In a further embodiment,
single-stranded RNAi (ssRNAi) compounds that act via the RNAi antisense
mechanism are contemplated.

[0039] Further modifications can be made to the double-stranded compounds
and may include conjugate groups attached to one of the termini, selected
nucleobase positions, sugar positions or to one of the internucleoside
linkages. Alternatively, the two strands can be linked via a non-nucleic
acid moiety or linker group. When formed from only one strand, dsRNA can
take the form of a self-complementary hairpin-type molecule that doubles
back on itself to form a duplex. Thus, the dsRNAs can be fully or
partially double-stranded. When formed from two strands, or a single
strand that takes the form of a self-complementary hairpin-type molecule
doubled back on itself to form a duplex, the two strands (or
duplex-forming regions of a single strand) are complementary RNA strands
that base pair in Watson-Crick fashion.

[0040] According to the present invention, "antisense compounds" include
antisense oligonucleotides, ribozymes, external guide sequence (EGS)
oligonucleotides, siRNA compounds, single- or double-stranded RNA
interference (RNAi) compounds such as siRNA compounds, and other
oligomeric compounds which hybridize to at least a portion of the target
nucleic acid and modulate its function. As such, they may be DNA, RNA,
DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or
more of these. These compounds may be single-stranded, double-stranded,
circular or hairpin oligomeric compounds and may contain structural
elements such as internal or terminal bulges, mismatches or loops.
Antisense compounds are routinely prepared linearly but can be joined or
otherwise prepared to be circular and/or branched. Antisense compounds
can include constructs such as, for example, two strands hybridized to
form a wholly or partially double-stranded compound or a single strand
with sufficient self-complementarity to allow for hybridization and
formation of a fully or partially double-stranded compound. The two
strands can be linked internally leaving free 3' or 5' termini or can be
linked to form a continuous hairpin structure or loop. The hairpin
structure may contain an overhang on either the 5' or 3' terminus
producing an extension of single stranded character. The double stranded
compounds optionally can include overhangs on the ends. Further
modifications can include conjugate groups attached to one of the
termini, selected nucleobase positions, sugar positions or to one of the
internucleoside linkages. Alternatively, the two strands can be linked
via a non-nucleic acid moiety or linker group. When formed from only one
strand, dsRNA can take the form of a self-complementary hairpin-type
molecule that doubles back on itself to form a duplex. Thus, the dsRNAs
can be fully or partially double stranded. Specific inhibition of gene
expression can be achieved by stable expression of dsRNA hairpins in
transgenic cell lines (Hammond et al., Nat. Rev. Genet., 1991, 2,
110-119; Matzke et al., Curr. Opin. Genet. Dev., 2001, 11, 221-227;
Sharp, Genes Dev., 2001, 15, 485-490). When formed from two strands, or a
single strand that takes the form of a self-complementary hairpin-type
molecule doubled back on itself to form a duplex, the two strands (or
duplex-forming regions of a single strand) are complementary RNA strands
that base pair in Watson-Crick fashion.

[0041] Once introduced to a system, the compounds of the invention may
elicit the action of one or more enzymes or structural proteins to effect
cleavage or other modification of the target nucleic acid or may work via
occupancy-based mechanisms. In general, nucleic acids (including
oligonucleotides) may be described as "DNA-like" (i.e., generally having
one or more 2'-deoxy sugars and, generally, T rather than U bases) or
"RNA-like" (i.e., generally having one or more 2'-hydroxyl or 2'-modified
sugars and, generally U rather than T bases). Nucleic acid helices can
adopt more than one type of structure, most commonly the A- and B-forms.
It is believed that, in general, oligonucleotides which have B-form-like
structure are "DNA-like" and those which have A-form-like structure are
"RNA-like." In some (chimeric) embodiments, an antisense compound may
contain both A- and B-form regions.

[0042] One example of an enzyme that modifies the target nucleic acid is
RNAse H, a cellular endonuclease which cleaves the RNA strand of an
RNA:DNA duplex. It is known in the art that single-stranded antisense
compounds which contain "DNA-like" regions (e.g., 2'-deoxy regions)
longer than about 3 or 4 consecutive nucleobases are able to recruit
RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA
target, thereby greatly enhancing the efficiency of
oligonucleotide-mediated inhibition of gene expression. More recently, a
dsRNAse has been postulated to be involved in the cleavage of the RNA
strand in the RNA:RNA duplex observed in the RNA interference (RNAi)
process.

[0043] While one well accepted form of antisense compound is a
single-stranded antisense oligonucleotide, in other contexts,
double-stranded RNA or analogs thereof are useful. In many species the
introduction of double-stranded structures, such as double-stranded RNA
(dsRNA) molecules, has been shown to induce potent and specific
antisense-mediated reduction of the function of a gene or its associated
gene products. This phenomenon occurs in both plants and animals and is
believed to have an evolutionary connection to viral defense and
transposon silencing (Guo et al., Cell, 1995, 81, 611-620; Montgomery et
al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The
posttranscriptional antisense mechanism defined in Caenorhabditis elegans
resulting from exposure to double-stranded RNA (dsRNA) has since been
designated RNA interference (RNAi). This term has been generalized to
mean antisense-mediated gene silencing involving the introduction of
dsRNA leading to the sequence-specific reduction of endogenous targeted
mRNA levels (Fire et al., Nature, 1998, 391, 806-811). The RNAi compounds
are often referred to as short interfering RNAs or siRNAs. Recently, it
has been shown that it is, in fact, the single-stranded RNA oligomers of
antisense polarity of the dsRNAs which are the potent inducers of RNAi
(Tijsterman et al., Science, 2002, 295, 694-697). Both RNAi compounds
(I.e., single- or double-stranded RNA or RNA-like compounds) and
single-stranded RNase H-dependent antisense compounds bind to their RNA
target by base pairing (i.e., hybridization) and induce site-specific
cleavage of the target RNA by specific RNAses; i.e., both work via an
antisense mechanism. Vickers et al., J. Biol. Chem., 2003, 278,
7108-7118.

[0044] In the context of this invention, the term "oligonucleotide" refers
to an oligomer or polymer of ribonucleic acid (RNA) and/or
deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog
thereof. This term includes oligonucleotides composed of naturally
occurring nucleobases, sugars and covalent internucleoside (backbone)
linkages as well as oligonucleotides having non-naturally occurring
portions which function similarly. Such modified or substituted
oligonucleotides are often desired over native forms because of desirable
properties such as, for example, enhanced cellular uptake, enhanced
affinity for a target nucleic acid and increased stability in the
presence of nucleases.

[0047] In one embodiment, the antisense compounds of the invention have
antisense portions of 12 or 13 to 30 nucleobases in length. One having
ordinary skill in the art will appreciate that this embodies antisense
compounds having antisense portions of 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases in length, or
any range therewithin.

[0048] In some embodiments, the antisense compounds of the invention have
antisense portions of 12 or 13 to 24 nucleobases in length. One having
ordinary skill in the art will appreciate that this embodies antisense
compounds having antisense portions of 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23 or 24 nucleobases in length, or any range therewithin.

[0049] In some embodiments, the antisense compounds of the invention have
antisense portions of 19 to 23 nucleobases in length. One having ordinary
skill in the art will appreciate that this embodies antisense compounds
having antisense portions of 19, 20, 21, 22 or 23 nucleobases in length,
or any range therewithin.

[0050] Antisense compounds 8-80 nucleobases in length comprising a stretch
of at least eight (8) consecutive nucleobases selected from within the
illustrative antisense compounds are considered to be suitable antisense
compounds as well.

[0051] Exemplary compounds include oligonucleotide sequences that comprise
at least the 8 consecutive nucleobases from the 5'-terminus of one of the
illustrative antisense compounds (the remaining nucleobases being a
consecutive stretch of the same oligonucleotide beginning immediately
upstream of the 5'-terminus of the antisense compound which is
specifically hybridizable to the target nucleic acid and continuing until
the oligonucleotide contains about 8 to about 80 nucleobases). Other
compounds are represented by oligonucleotide sequences that comprise at
least the 8 consecutive nucleobases from the 3'-terminus of one of the
illustrative antisense compounds (the remaining nucleobases being a
consecutive stretch of the same oligonucleotide beginning immediately
downstream of the 3'-terminus of the antisense compound which is
specifically hybridizable to the target nucleic acid and continuing until
the oligonucleotide contains about 8 to about 80 nucleobases). It is also
understood that compounds may be represented by oligonucleotide sequences
that comprise at least 8 consecutive nucleobases from an internal portion
of the sequence of an illustrative compound, and may extend in either or
both directions until the oligonucleotide contains about 8 about 80
nucleobases.

[0052] It should be noted that oligomeric compounds or pharmaceutically
acceptable salts thereof of the present invention do not include the
nucleobase sequence 5'-AGTCGCCATCTTAGATCGAT-3' (SEQ ID NO:454) or
5'-AGUCGCCAUCUUAGAUCGAU-3' (SEQ ID NO:455). Furthermore, oligomeric
compounds or pharmaceutically acceptable salts thereof encompassed by the
present invention can consist of, consist essentially of, or comprise,
the specific nucleotide sequences disclosed herein. The phrases "consist
essentially of," "consists essentially of," "consisting essentially of,"
or the like when applied to oligomeric compounds or pharmaceutically
acceptable salts thereof encompassed by the present invention refer to
nucleotide sequences like those disclosed herein, but which contain
additional nucleotides (ribonucleotides, deoxyribonucleotides, or analogs
or derivatives thereof as discussed herein). Such additional nucleotides,
however, do not materially affect the basic and novel characteristic(s)
of these oligomeric compounds or pharmaceutically acceptable salts
thereof in modulating, attenuating, or inhibiting eIF4E gene expression
or RNA function, including the specific quantitative effects of these
molecules, compared to the corresponding parameters of the corresponding
oligomeric compounds or pharmaceutically acceptable salts thereof
disclosed herein.

[0053] One having skill in the art armed with the antisense compounds
illustrated herein will be able, without undue experimentation, to
identify further antisense compounds.

C. Targets of the Invention

[0054] "Targeting" an oligomeric compound to a particular nucleic acid
molecule, in the context of this invention, can be a multistep process.
The process usually begins with the identification of a target nucleic
acid whose function is to be modulated. This target nucleic acid may be,
for example, a cellular gene (or mRNA transcribed from the gene) whose
expression is associated with a particular disorder or disease state, or
a nucleic acid molecule from an infectious agent. In the present
invention, the target nucleic acid encodes eIF4E.

[0055] The targeting process usually also includes determination of at
least one target region, segment, or site within the target nucleic acid
for the antisense interaction to occur such that the desired effect,
e.g., modulation of expression, will result. Within the context of the
present invention, the term "region" is defined as a portion of the
target nucleic acid having at least one identifiable structure, function,
or characteristic. Within regions of target nucleic acids are segments.
"Segments" are defined as smaller or sub-portions of regions within a
target nucleic acid. "Sites," as used in the present invention, are
defined as positions within a target nucleic acid.

[0056] Since, as is known in the art, the translation initiation codon is
typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the
corresponding DNA molecule), the translation initiation codon is also
referred to as the "AUG codon," the "start codon" or the "AUG start
codon." A minority of genes have a translation initiation codon having
the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG
have been shown to function in vivo. Thus, the terms "translation
initiation codon" and "start codon" can encompass many codon sequences,
even though the initiator amino acid in each instance is typically
methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is
also known in the art that eukaryotic and prokaryotic genes may have two
or more alternative start codons, any one of which may be preferentially
utilized for translation initiation in a particular cell type or tissue,
or under a particular set of conditions. In the context of the invention,
"start codon" and "translation initiation codon" refer to the codon or
codons that are used in vivo to initiate translation of an mRNA
transcribed from a gene encoding eIF4E, regardless of the sequence(s) of
such codons. It is also known in the art that a translation termination
codon (or "stop codon") of a gene may have one of three sequences, i.e.,
5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA,
5'-TAG and 5'-TGA, respectively).

[0057] The terms "start codon region" and "translation initiation codon
region" refer to a portion of such an mRNA or gene that encompasses from
about 25 to about 50 contiguous nucleotides in either direction (i.e., 5'
or 3') from a translation initiation codon. Similarly, the terms "stop
codon region" and "translation termination codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to about
50 contiguous nucleotides in either direction (i.e., 5' or 3') from a
translation termination codon. Consequently, the "start codon region" (or
"translation initiation codon region") and the "stop codon region" (or
"translation termination codon region") are all regions which may be
targeted effectively with the antisense compounds of the present
invention.

[0058] The open reading frame (ORF) or "coding region," which is known in
the art to refer to the region between the translation initiation codon
and the translation termination codon, is also a region which may be
targeted effectively. Within the context of the present invention, one
region is the intragenic region encompassing the translation initiation
or termination codon of the open reading frame (ORF) of a gene.

[0059] Other target regions include the 5' untranslated region (5'UTR),
known in the art to refer to the portion of an mRNA in the 5' direction
from the translation initiation codon, and thus including nucleotides
between the 5' cap site and the translation initiation codon of an mRNA
(or corresponding nucleotides on the gene), and the 3' untranslated
region (3'UTR), known in the art to refer to the portion of an mRNA in
the 3' direction from the translation termination codon, and thus
including nucleotides between the translation termination codon and 3'
end of an mRNA (or corresponding nucleotides on the gene). The 5' cap
site of an mRNA comprises an N7-methylated guanosine residue joined to
the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5'
cap region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap site. The
5' cap region is also a target.

[0060] Although some eukaryotic mRNA transcripts are directly translated,
many contain one or more regions, known as "introns," which are excised
from a transcript before it is translated. The remaining (and therefore
translated) regions are known as "exons" and are spliced together to form
a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon
junctions or exon-intron junctions, may also be particularly useful in
situations where aberrant splicing is implicated in disease, or where an
overproduction of a particular splice product is implicated in disease.
Aberrant fusion junctions due to rearrangements or deletions are also
suitable target sites. mRNA transcripts produced via the process of
splicing of two (or more) mRNAs from different gene sources are known as
"fusion transcripts." It is also known that introns can be effectively
targeted using antisense compounds targeted to, for example, DNA or
pre-mRNA. Single-stranded antisense compounds such as oligonucleotide
compounds that work via an RNase H mechanism are effective for targeting
pre-mRNA.

[0061] It is also known in the art that alternative RNA transcripts can be
produced from the same genomic region of DNA. These alternative
transcripts are generally known as "variants." More specifically,
"pre-mRNA variants" are transcripts produced from the same genomic DNA
that differ from other transcripts produced from the same genomic DNA in
either their start or stop position and contain both intronic and exonic
sequence.

[0062] Upon excision of one or more exon or intron regions, or portions
thereof during splicing, pre-mRNA variants produce smaller "mRNA
variants." Consequently, mRNA variants are processed pre-mRNA variants
and each unique pre-mRNA variant must always produce a unique mRNA
variant as a result of splicing. These mRNA variants are also known as
"alternative splice variants." If no splicing of the pre-mRNA variant
occurs then the pre-mRNA variant is identical to the mRNA variant.

[0063] It is also known in the art that variants can be produced through
the use of alternative signals to start or stop transcription and that
pre-mRNAs and mRNAs can possess more that one start codon or stop codon.
Variants that originate from a pre-mRNA or mRNA that use alternative
start codons are known as "alternative start variants" of that pre-mRNA
or mRNA. Those transcripts that use an alternative stop codon are known
as "alternative stop variants" of that pre-mRNA or mRNA. One specific
type of alternative stop variant is the "polyA variant" in which the
multiple transcripts produced result from the alternative selection of
one of the "polyA stop signals" by the transcription machinery, thereby
producing transcripts that terminate at unique polyA sites. Within the
context of the invention, the types of variants described herein are also
suitable target nucleic acids.

[0064] The locations on the target nucleic acid to which the suitable
oligomeric compounds hybridize are hereinbelow referred to as "suitable
target segments." As used herein the term "suitable target segment" is
defined as at least an 8-nucleobase portion of a target region to which
an active oligomeric compound is targeted. While not wishing to be bound
by theory, it is presently believed that these target segments represent
portions of the target nucleic acid which are accessible for
hybridization.

[0065] While the specific sequences of certain suitable target segments
are set forth herein, one of skill in the art will recognize that these
serve to illustrate and describe particular embodiments within the scope
of the present invention. Additional suitable target segments may be
identified by one having ordinary skill. It is not necessary that the
"suitable target segment" be identified by this term or included in a
"suitable target segment" table, if any.

[0066] Target segments 8-80 nucleobases in length comprising a stretch of
at least eight (8) consecutive nucleobases selected from within the
illustrative suitable target segments are considered to be suitable for
targeting as well.

[0067] Target segments can include DNA or RNA sequences that comprise at
least the 8 consecutive nucleobases from the 5'-terminus of one of the
illustrative suitable target segments (the remaining nucleobases being a
consecutive stretch of the same DNA or RNA beginning immediately upstream
of the 5'-terminus of the target segment and continuing until the DNA or
RNA contains about 8 to about 80 nucleobases). Similarly suitable target
segments are represented by DNA or RNA sequences that comprise at least
the 8 consecutive nucleobases from the 3'-terminus of one of the
illustrative suitable target segments (the remaining nucleobases being a
consecutive stretch of the same DNA or RNA beginning immediately
downstream of the 3'-terminus of the target segment and continuing until
the DNA or RNA contains about 8 to about 80 nucleobases). It is also
understood that suitable oligomeric target segments may be represented by
DNA or RNA sequences that comprise at least 8 consecutive nucleobases
from an internal portion of the sequence of an illustrative suitable
target segments, and may extend in either or both directions until the
oligonucleotide contains about 8 about 80 nucleobases. One having skill
in the art armed with the suitable target segments illustrated herein
will be able, without undue experimentation, to identify further suitable
target segments.

[0068] Once one or more target regions, segments or sites have been
identified, oligomeric compounds are chosen which are sufficiently
complementary to the target, i.e., hybridize sufficiently well and with
sufficient specificity, to give the desired effect.

[0070] In a further embodiment, the "suitable target segments" identified
herein may be employed in a screen for additional compounds that modulate
the expression of eIF4E. "Modulators" are those compounds that decrease
or increase the expression of a nucleic acid molecule encoding eIF4E and
which comprise at least an 8-nucleobase portion which is complementary
(i.e., antisense) to a suitable target segment. The screening method
comprises the steps of contacting a suitable target segment of a nucleic
acid molecule encoding eIF4E with one or more candidate modulators, and
selecting for one or more candidate modulators which decrease or increase
the expression of a nucleic acid molecule encoding eIF4E. Once it is
shown that the candidate modulator or modulators are capable of
modulating (e.g. either decreasing or increasing) the expression of a
nucleic acid molecule encoding eIF4E, the modulator may then be employed
in further investigative studies of the function of eIF4E, or for use as
a research, diagnostic, or therapeutic agent in accordance with the
present invention.

[0072] The oligomeric compounds of the present invention can also be
applied in the areas of drug discovery and target validation. The present
invention comprehends the use of the compounds and suitable target
segments identified herein in drug discovery efforts to elucidate
relationships that exist between eIF4E and a disease state, phenotype, or
condition. These methods include detecting or modulating eIF4E comprising
contacting a sample, tissue, cell, or organism with one or more antisense
compounds of the present invention, measuring the nucleic acid or protein
level of eIF4E and/or a related phenotypic or chemical endpoint at some
time after treatment, and optionally comparing the measured value to a
non-treated sample or sample treated with a further compound of the
invention. These methods can also be performed in parallel or in
combination with other experiments to determine the function of unknown
genes for the process of target validation or to determine the validity
of a particular gene product as a target for treatment or prevention of a
particular disease, condition, or phenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0073] The oligomeric compounds of the present invention can be utilized
for diagnostics, therapeutics, prophylaxis and as research reagents and
kits. Furthermore, oligomeric compounds, which are able to inhibit gene
expression with exquisite specificity, are often used by those of
ordinary skill to elucidate the function of particular genes or to
distinguish between functions of various members of a biological pathway.

[0074] For use in kits and diagnostics, the compounds of the present
invention, either alone or in combination with other compounds or
therapeutics, can be used as tools in differential and/or combinatorial
analyses to elucidate expression patterns of a portion or the entire
complement of genes expressed within cells and tissues.

[0075] As one nonlimiting example, expression patterns within cells or
tissues treated with one or more compounds are compared to control cells
or tissues not treated with compounds and the patterns produced are
analyzed for differential levels of gene expression as they pertain, for
example, to disease association, signaling pathway, cellular
localization, expression level, size, structure or function of the genes
examined. These analyses can be performed on stimulated or unstimulated
cells and in the presence or absence of other compounds which affect
expression patterns.

[0077] The specificity and sensitivity of antisense is also harnessed by
those of skill in the art for therapeutic uses. Antisense compounds have
been employed as therapeutic moieties in the treatment of disease states
in animals, including humans. Antisense drugs, including ribozymes, have
been safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that antisense
compounds are useful therapeutic modalities that can be configured to be
useful in treatment regimes for the treatment of cells, tissues and
animals, especially humans. Treatment of animals selected from companion,
zoo, and farm animals, including, but not limited to, cats, dogs,
rodents, horses, cows, sheep, pigs, goats, etc. is contemplated by the
present invention.

[0078] For therapeutics, an animal, such as a human, suspected of having a
disease or disorder which can be treated by modulating the expression of
eIF4E is treated by administering compounds in accordance with this
invention. For example, in one non-limiting embodiment, the methods
comprise the step of administering to the animal in need of treatment, a
therapeutically effective amount of an oligomeric compound that inhibits
eIF4E. The eIF4E compounds of the present invention effectively inhibit
the activity or expression of a nucleic acid encoding eIF4E RNA. Because
reduction in eIF4E RNA levels can lead to reduction in eIF4E protein
levels as well, reduction in protein expression or levels can also be
measured. In some embodiments, the animal is diagnosed for the disease or
disorder prior to treatment. In one embodiment, the oligomeric compounds
modulate the activity or expression of eIF4E mRNA by at least about 10%,
by at least about 20%, by at least about 25%, by at least about 30%, by
at least about 40%, by at least about 50%, by at least about 60%, by at
least about 70%, by at least about 75%, by at least about 80%, by at
least about 85%, by at least about 90%, by at least about 95%, by at
least about 98%, by at least about 99%, or by 100%.

[0079] For example, the reduction of the expression of eIF4E may be
measured in serum, adipose tissue, liver or any other body fluid, tissue
or organ of the animal. The cells contained within said fluids, tissues
or organs being analyzed can contain a nucleic acid molecule encoding
eIF4E protein and/or the eIF4E protein itself.

[0080] The compounds of the invention can be utilized in pharmaceutical
compositions by adding an effective amount of a compound to a suitable
pharmaceutically or physiologically acceptable excipient, diluent or
carrier. Use of the compounds and methods of the invention may also be
useful prophylactically. Thus, the present invention encompasses the use
of the compounds disclosed herein as pharmaceuticals, as well as the use
of the presently disclosed compounds for the preparation of medicaments
for the treatment of disorders as disclosed herein.

[0081] The compounds of the present invention inhibit the expression of
eIF4E. Because these compounds inhibit the effects of eIF4E activation,
the compounds are useful in the treatment of disorders related to eIF4E
expression. Thus, the compounds of the present invention are
antineoplastic agents.

[0083] Thus, in one embodiment, the present invention provides a method
for the treatment of susceptible neoplasms comprising administering to a
patient in need thereof an effective amount of an isolated single
stranded RNA or double stranded RNA oligonucleotide directed to eIF4E.
The ssRNA or dsRNA oligonucleotide may be modified or unmodified. That
is, the present invention provides for the use of an isolated double
stranded RNA oligonucleotide targeted to eIF4E, or a pharmaceutical
composition thereof, for the treatment of susceptible neoplasms.

[0084] In another aspect, the present invention provides for the use of a
compound of an isolated double stranded RNA oligonucleotide in the
manufacture of a medicament for inhibiting eIF4E expression or
overexpression. Thus, the present invention provides for the use of an
isolated double stranded RNA oligonucleotide targeted to eIF4E in the
manufacture of a medicament for the treatment of susceptible neoplasms by
means of the method described above.

[0085] The compounds of the present invention are useful for the treatment
of hyperproliferative disorders. Specifically, the compounds of the
present invention are useful for the treatment of cancer. The compounds
of the present invention are particularly useful for the treatment of
solid tumors. Thus, the compounds of the present invention are especially
useful for the treatment of breast cancer, colon cancer, prostate cancer,
lung cancer, liver cancer, bladder cancer, ovarian cancer, renal cancer
and glioblastoma. The antisense compounds of the present invention are
particularly useful for the treatment of solid tumors.

F. Modifications

[0086] As is known in the art, a nucleoside is a base-sugar combination.
The base portion of the nucleoside is normally a heterocyclic base
(sometimes referred to as a "nucleobase" or simply a "base"). The two
most common classes of such heterocyclic bases are the purines and the
pyrimidines. Nucleotides are nucleosides that further include a phosphate
group covalently linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate group can
be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In
forming oligonucleotides, the phosphate groups covalently link adjacent
nucleosides to one another to form a linear polymeric compound. In turn,
the respective ends of this linear polymeric compound can be further
joined to form a circular compound, however, linear compounds are
generally desired. In addition, linear compounds may have internal
nucleobase complementarity and may therefore fold in a manner as to
produce a fully or partially double-stranded compound. Within
oligonucleotides, the phosphate groups are commonly referred to as
forming the internucleoside backbone of the oligonucleotide. The normal
linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Modified Sugar and Internucleoside Linkages

[0087] Specific examples of oligomeric antisense compounds useful in this
invention include oligonucleotides containing modified e.g. non-naturally
occurring internucleoside linkages. As defined in this specification,
oligonucleotides having modified internucleoside linkages include
internucleoside linkages that retain a phosphorus atom and
internucleoside linkages that do not have a phosphorus atom. For the
purposes of this specification, and as sometimes referenced in the art,
modified oligonucleotides that do not have a phosphorus atom in their
internucleoside backbone can also be considered to be oligonucleosides.

[0088] Oligomeric compounds of the invention can have one or more modified
internucleoside linkages. One phosphorus-containing modified
internucleoside linkage is the phosphorothioate internucleoside linkage.
Other modified oligonucleotide backbones containing a phosphorus atom
therein include, for example, phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotri-esters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral
phosphonates, phosphinates, phosphoramidates including 3'-amino
phosphoramidate and amino-alkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, phosphonoacetate
and thiophosphonoacetate (see Sheehan et al., Nucleic Acids Research,
2003, 31(14), 4109-4118 and Dellinger et al., J. Am. Chem. Soc., 2003,
125, 940-950), selenophosphates and borano-phosphates having normal 3'-5'
linkages, 2'-5' linked analogs of these, and those having inverted
polarity wherein one or more internucleotide linkages is a 3' to 3', 5'
to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity
comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage
i.e. a single inverted nucleoside residue which may be abasic (the
nucleobase is missing or has a hydroxyl group in place thereof). Various
salts, mixed salts and free acid forms are also included.

[0091] In some embodiments of the invention, oligomeric compounds may have
one or more phosphorothioate and/or heteroatom internucleoside linkages,
in particular --CH2--NH--O--CH2--,
--CH2--N(CH3)--O--CH2-- (known as a methylene
(methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--,
--CH2--N(CH3)--N(CH3)--CH2-- and
--O--N(CH3)--CH2--CH2-- (wherein the native phosphodiester
internucleotide linkage is represented as
--O--P(═O)(OH)--O--CH2--). The MMI type internucleoside linkages
are disclosed in the above referenced U.S. Pat. No. 5,489,677. Amide
internucleoside linkages are disclosed in the above referenced U.S. Pat.
No. 5,602,240.

[0092] Some oligonucleotide backbones that do not include a phosphorus
atom therein have backbones that are formed by short chain alkyl or
cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or
cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic internucleoside linkages. These include
those having morpholino linkages (formed in part from the sugar portion
of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone
backbones; formacetyl and thioformacetyl backbones; methylene formacetyl
and thioformacetyl backbones; riboacetyl backbones; alkene containing
backbones; sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones; and
others having mixed N, O, S and CH2 component parts.

[0094] Another group of oligomeric compounds amenable to the present
invention includes oligonucleotide mimetics. The term mimetic as it is
applied to oligonucleotides is intended to include oligomeric compounds
wherein the furanose ring or the furanose ring and the internucleotide
linkage are replaced with novel groups, replacement of only the furanose
ring is also referred to in the art as being a sugar surrogate. The
heterocyclic base moiety or a modified heterocyclic base moiety is
maintained for hybridization with an appropriate target nucleic acid.

[0095] One such oligomeric compound, an oligonucleotide mimetic that has
been shown to have excellent hybridization properties, is referred to as
a peptide nucleic acid (PNA). Nielsen et al., Science, 1991, 254,
1497-1500. PNAs have favorable hybridization properties, high biological
stability and are electrostatically neutral molecules. In one recent
study PNA compounds were used to correct aberrant splicing in a
transgenic mouse model (Sazani et al., Nat. Biotechnol., 2002, 20,
1228-1233). In PNA oligomeric compounds, the sugar-backbone of an
oligonucleotide is replaced with an amide containing backbone, in
particular an aminoethylglycine backbone. The nucleobases are bound
directly or indirectly (--C(═O)--CH2-- as shown below) to aza
nitrogen atoms of the amide portion of the backbone. Representative
United States patents that teach the preparation of PNA oligomeric
compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;
5,714,331; and 5,719,262, each of which is herein incorporated by
reference. PNA compounds can be obtained commercially from Applied
Biosystems (Foster City, Calif., USA). Numerous modifications to the
basic PNA backbone are known in the art; particularly useful are PNA
compounds with one or more amino acids conjugated to one or both termini.
In particular, 1-8 lysine or arginine residues are useful when conjugated
to the end of a PNA molecule.

[0096] Another class of oligonucleotide mimetic that has been studied is
based on linked morpholino units (morpholino nucleic acid) having
heterocyclic bases attached to the morpholino ring. A number of linking
groups have been reported that link the morpholino monomeric units in a
morpholino nucleic acid. One class of linking groups have been selected
to give a non-ionic oligomeric compound. The non-ionic morpholino-based
oligomeric compounds are less likely to have undesired interactions with
cellular proteins. Morpholino-based oligomeric compounds are non-ionic
mimics of oligonucleotides which are less likely to form undesired
interactions with cellular proteins (Braasch et al., Biochemistry, 2002,
41(14), 4503-4510). Morpholino-based oligomeric compounds have been
studied in zebrafish embryos (see: Genesis, volume 30, issue 3, 2001 and
Heasman, J., Dev. Biol., 2002, 243, 209-214). Further studies of
morpholino-based oligomeric compounds have also been reported (see:
Nasevicius et al., Nat. Genet., 2000, 26, 216-220; and Lacerra et al.,
Proc. Natl. Acad. Sci., 2000, 97, 9591-9596). Morpholino-based oligomeric
compounds are disclosed in U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.
The morpholino class of oligomeric compounds have been prepared having a
variety of different linking groups joining the monomeric subunits.
Linking groups can be varied from chiral to achiral, and from charged to
neutral. U.S. Pat. No. 5,166,315 discloses linkages including
--O--P(═O)(N(CH3)2)--O--; U.S. Pat. No. 5,034,506 discloses
achiral intermorpholino linkages; and U.S. Pat. No. 5,185,444 discloses
phosphorus containing chiral intermorpholino linkages.

[0097] A further class of oligonucleotide mimetic is referred to as
cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in
a DNA or RNA molecule is replaced with a cyclohenyl ring. CeNA DMT
protected phosphoramidite monomers have been prepared and used for
oligomeric compound synthesis following classical phosphoramidite
chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides
having specific positions modified with CeNA have been prepared and
studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In
general the incorporation of CeNA monomers into a DNA chain increases its
stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with
RNA and DNA complements with similar stability to the native complexes.
The study of incorporating CeNA structures into natural nucleic acid
structures was shown by NMR and circular dichroism to proceed with easy
conformational adaptation. Furthermore the incorporation of CeNA into a
sequence targeting RNA was stable to serum and able to activate E. coli
RNase resulting in cleavage of the target RNA strand.

[0098] A further modification includes bicyclic sugar moieties such as
"Locked Nucleic Acids" (LNAs) in which the 2'-hydroxyl group of the
ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring
thereby forming a 2'-C,4'-C-oxymethylene linkage to form the bicyclic
sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs,
2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et
al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos.
6,268,490 and 6,670,461). The linkage can be a methylene (--CH2--)
group bridging the 2' oxygen atom and the 4' carbon atom, for which the
term LNA is used for the bicyclic moiety; in the case of an ethylene
group in this position, the term ENA® is used (Singh et al., Chem.
Commun., 1998, 4, 455-456; ENA®: Morita et al., Bioorganic Medicinal
Chemistry, 2003, 11, 2211-2226). LNA and other bicyclic sugar analogs
display very high duplex thermal stabilities with complementary DNA and
RNA (Tm=+3 to +10 C), stability towards 3'-exonucleolytic degradation and
good solubility properties. LNAs are commercially available from ProLigo
(Paris, France and Boulder, Colo., USA).

[0099] An isomer of LNA that has also been studied is V-L-LNA which has
been shown to have superior stability against a 3'-exonuclease (Frieden
et al., Nucleic Acids Research, 2003, 21, 6365-6372). The V-L-LNAs were
incorporated into antisense gapmers and chimeras that showed potent
antisense activity.

[0100] Another similar bicyclic sugar moiety that has been prepared and
studied has the bridge going from the 3'-hydroxyl group via a single
methylene group to the 4' carbon atom of the sugar ring thereby forming a
3'-C,4'-C-oxymethylene linkage (see U.S. Pat. No. 6,043,060).

[0101] The conformations of LNAs determined by 2D NMR spectroscopy have
shown that the locked orientation of the LNA nucleotides, both in
single-stranded LNA and in duplexes, constrains the phosphate backbone in
such a way as to introduce a higher population of the N-type conformation
(Petersen et al., J. Mol. Recognit., 2000, 13, 44-53). These
conformations are associated with improved stacking of the nucleobases
(Wengel et al., Nucleosides Nucleotides, 1999, 18, 1365-1370).

[0102] LNA has been shown to form exceedingly stable LNA:LNA duplexes
(Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA
hybridization was shown to be the most thermally stable nucleic acid type
duplex system, and the RNA-mimicking character of LNA was established at
the duplex level. Introduction of 3 LNA monomers (T or A) significantly
increased melting points (Tm=+15/+11) toward DNA complements. The
universality of LNA-mediated hybridization has been stressed by the
formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of
LNA was reflected with regard to the N-type conformational restriction of
the monomers and to the secondary structure of the LNA:RNA duplex.

[0105] The synthesis and preparation of the LNA monomers adenine,
cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with
their oligomerization, and nucleic acid recognition properties have been
described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and
preparation thereof are also described in WO 98/39352 and WO 99/14226.

[0106] The first analogs of LNA, phosphorothioate-LNA and 2'-thio-LNAs,
have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,
2219-2222). Preparation of locked nucleoside analogs containing
oligodeoxyribonucleotide duplexes as substrates for nucleic acid
polymerases has also been described (Wengel et al., WO 99/14226).
Furthermore, synthesis of 2'-amino-LNA, a novel conformationally
restricted high-affinity oligonucleotide analog has been described in the
art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,
2'-Amino- and 2'-methylamino-LNA's have been prepared and the thermal
stability of their duplexes with complementary RNA and DNA strands has
been previously reported.

[0107] Another oligonucleotide mimetic amenable to the present invention
that has been prepared and studied is threose nucleic acid. This
oligonucleotide mimetic is based on threose nucleosides instead of ribose
nucleosides. Initial interest in (3',2')-.A-inverted.-L-threose nucleic
acid (TNA) was directed to the question of whether a DNA polymerase
existed that would copy the TNA. It was found that certain DNA
polymerases are able to copy limited stretches of a TNA template
(reported in C&EN/Jan. 13, 2003). In another study it was determined that
TNA is capable of antiparallel Watson-Crick base pairing with
complementary DNA, RNA and TNA oligonucleotides (Chaput et al., J. Am.
Chem. Soc., 2003, 125, 856-857).

[0108] In one study (3',2')-.A-inverted.-L-threose nucleic acid was
prepared and compared to the 2' and 3' amidate analogs (Wu et al.,
Organic Letters, 2002, 4(8), 1279-1282). The amidate analogs were shown
to bind to RNA and DNA with comparable strength to that of RNA/DNA.

[0110] Another class of oligonucleotide mimetic is referred to as
phosphonomonoester nucleic acids which incorporate a phosphorus group in
the backbone. This class of olignucleotide mimetic is reported to have
useful physical and biological and pharmacological properties in the
areas of inhibiting gene expression (antisense oligonucleotides,
ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),
as probes for the detection of nucleic acids and as auxiliaries for use
in molecular biology. Further oligonucleotide mimetics amenable to the
present invention have been prepared wherein a cyclobutyl ring replaces
the naturally occurring furanosyl ring.

[0111] Oligomeric compounds may also contain one or more substituted sugar
moieties. Suitable compounds can comprise one of the following at the 2'
position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or
N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may
be substituted or unsubstituted C1 to C10 alkyl or C2 to
C10 alkenyl and alkynyl. Particularly suitable are
O((CH2)nO)mCH3, O(CH2)nOCH3,
O(CH2)nNH2, O(CH2)nCH3,
O(CH2)nONH2, and
O(CH2)nON((CH2)nCH3)2, where n and m are
from 1 to about 10. Other oligonucleotides comprise one of the following
at the 2' position: C1 to C10 lower alkyl, substituted lower
alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,
SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3,
SO2CH3, ONO2, NO2, N3, NH2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,
substituted silyl, an RNA cleaving group, a reporter group, an
intercalator, a group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic properties
of an oligonucleotide, and other substituents having similar properties.
One modification includes 2'-methoxyethoxy
(2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or
2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an
alkoxyalkoxy group. A further modification includes
2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2
group, also known as 2'-DMAOE, as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH2--O--CH2--N(CH3)2, also described in
examples hereinbelow.

[0112] Other modifications include 2'-methoxy (2'-O--CH3),
2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl
(2'-CH2--CH═CH2), 2'-O-allyl
(2'-O--CH2--CH═CH2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. One 2'-arabino modification is 2'-F. Similar modifications may
also be made at other positions on the oligonucleotide, particularly the
3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked
oligonucleotides and the 5' position of 5' terminal nucleotide. Antisense
compounds may also have sugar mimetics such as cyclobutyl moieties in
place of the pentofuranosyl sugar. Representative United States patents
that teach the preparation of such modified sugar structures include, but
are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920,
each of which is herein incorporated by reference in its entirety.

[0113] In one aspect of the present invention oligomeric compounds include
nucleosides synthetically modified to induce a 3'-endo sugar
conformation. A nucleoside can incorporate synthetic modifications of the
heterocyclic base, the sugar moiety or both to induce a desired 3'-endo
sugar conformation. These modified nucleosides are used to mimic RNA like
nucleosides so that particular properties of an oligomeric compound can
be enhanced while maintaining the desirable 3'-endo conformational
geometry. There is an apparent preference for an RNA type duplex (A form
helix, predominantly 3'-endo) as a requirement (e.g. trigger) of RNA
interference which is supported in part by the fact that duplexes
composed of 2'-deoxy-2'-F-nucleosides appears efficient in triggering
RNAi response in the C. elegans system. Properties that are enhanced by
using more stable 3'-endo nucleosides include but are not limited to:
modulation of pharmacokinetic properties through modification of protein
binding, protein off-rate, absorption and clearance; modulation of
nuclease stability as well as chemical stability; modulation of the
binding affinity and specificity of the oligomer (affinity and
specificity for enzymes as well as for complementary sequences); and
increasing efficacy of RNA cleavage. The present invention provides
oligomeric triggers of RNAi having one or more nucleosides modified in
such a way as to favor a C3'-endo type conformation.

[0116] One conformation of modified nucleosides and their oligomers can be
estimated by various methods such as molecular dynamics calculations,
nuclear magnetic resonance spectroscopy and CD measurements. Hence,
modifications predicted to induce RNA like conformations, A-form duplex
geometry in an oligomeric context, are selected for use in the modified
oligonucleotides of the present invention. The synthesis of numerous of
the modified nucleosides amenable to the present invention are known in
the art (see for example, Chemistry of Nucleosides and Nucleotides Vol
1-3, ed. Leroy B. Townsend, 1988, Plenum press., and the examples section
below.)

[0117] The terms used to describe the conformational geometry of
homoduplex nucleic acids are "A Form" for RNA and "B Form" for DNA. The
respective conformational geometry for RNA and DNA duplexes was
determined from X-ray diffraction analysis of nucleic acid fibers (Arnott
and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general,
RNA:RNA duplexes are more stable and have higher melting temperatures
(Tms) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid
Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,
Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,
1997, 25, 2627-2634). The increased stability of RNA has been attributed
to several structural features, most notably the improved base stacking
interactions that result from an A-form geometry (Searle et al., Nucleic
Acids Res., 1993, 21, 2051-2056). The presence of the 2' hydroxyl in RNA
biases the sugar toward a C3' endo pucker, i.e., also designated as
Northern pucker, which causes the duplex to favor the A-form geometry. In
addition, the 2' hydroxyl groups of RNA can form a network of water
mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,
Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic
acids prefer a C2' endo sugar pucker, i.e., also known as Southern
pucker, which is thought to impart a less stable B-form geometry (Sanger,
W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New
York, N.Y.). As used herein, B-form geometry is inclusive of both
C2'-endo pucker and O4'-endo pucker. This is consistent with Berger, et.
al., Nucleic Acids Research, 1998, 26, 2473-2480, who pointed out that in
considering the furanose conformations which give rise to B-form duplexes
consideration should also be given to a O4'-endo pucker contribution.

[0118] DNA:RNA hybrid duplexes, however, are usually less stable than pure
RNA:RNA duplexes, and depending on their sequence may be either more or
less stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,
1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate
between A- and B-form geometries, which may result in poor stacking
interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff
et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry,
1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533).
The stability of the duplex formed between a target RNA and a synthetic
sequence is central to therapies such as but not limited to antisense and
RNA interference as these mechanisms require the binding of a synthetic
oligomer strand to an RNA target strand. In the case of antisense,
effective inhibition of the mRNA requires that the antisense DNA have a
very high binding affinity with the mRNA. Otherwise the desired
interaction between the synthetic oligomer strand and target mRNA strand
will occur infrequently, resulting in decreased efficacy.

[0119] One routinely used method of modifying the sugar puckering is the
substitution of the sugar at the 2'-position with a substituent group
that influences the sugar geometry. The influence on ring conformation is
dependant on the nature of the substituent at the 2'-position. A number
of different substituents have been studied to determine their sugar
puckering effect. For example, 2'-halogens have been studied showing that
the 2'-fluoro derivative exhibits the largest population (65%) of the
C3'-endo form, and the 2'-iodo exhibits the lowest population (7%). The
populations of adenosine (2'-OH) versus deoxyadenosine (2'-H) are 36% and
19%, respectively. Furthermore, the effect of the 2'-fluoro group of
adenosine dimers
(2'-deoxy-2'-fluoroadenosine-2'-deoxy-2'-fluoro-adenosine) is further
correlated to the stabilization of the stacked conformation.

[0120] As expected, the relative duplex stability can be enhanced by
replacement of 2'-OH groups with 2'-F groups thereby increasing the
C3'-endo population. It is assumed that the highly polar nature of the
2'-F bond and the extreme preference for C3'-endo puckering may stabilize
the stacked conformation in an A-form duplex. Data from UV
hypochromicity, circular dichroism, and 1H NMR also indicate that
the degree of stacking decreases as the electronegativity of the halo
substituent decreases. Furthermore, steric bulk at the 2'-position of the
sugar moiety is better accommodated in an A-form duplex than a B-form
duplex. Thus, a 2'-substituent on the 3'-terminus of a dinucleoside
monophosphate is thought to exert a number of effects on the stacking
conformation: steric repulsion, furanose puckering preference,
electrostatic repulsion, hydrophobic attraction, and hydrogen bonding
capabilities. These substituent effects are thought to be determined by
the molecular size, electronegativity, and hydrophobicity of the
substituent. Melting temperatures of complementary strands is also
increased with the 2'-substituted adenosine diphosphates. It is not clear
whether the 3'-endo preference of the conformation or the presence of the
substituent is responsible for the increased binding. However, greater
overlap of adjacent bases (stacking) can be achieved with the 3'-endo
conformation.

[0121] Increasing the percentage of C3'-endo sugars in a modified
oligonucleotide targeted to an RNA target strand should preorganize this
strand for binding to RNA. Of the several sugar modifications that have
been reported and studied in the literature, the incorporation of
electronegative substituents such as 2'-fluoro or 2'-alkoxy shift the
sugar conformation towards the 3' endo (northern) pucker conformation.
This preorganizes an oligonucleotide that incorporates such modifications
to have an A-form conformational geometry. This A-form conformation
results in increased binding affinity of the oligonucleotide to a target
RNA strand.

[0122] Representative 2'-substituent groups amenable to the present
invention that give A-form conformational properties (3'-endo) to the
resultant duplexes include 2'-O-alkyl, 2'-O-substituted alkyl and
2'-fluoro substituent groups. Suitable for the substituent groups are
various alkyl and aryl ethers and thioethers, amines and monoalkyl and
dialkyl substituted amines. It is further intended that multiple
modifications can be made to one or more of the oligomeric compounds of
the invention at multiple sites of one or more monomeric subunits
(nucleosides are suitable) and or internucleoside linkages to enhance
properties such as but not limited to activity in a selected application.

Natural and Modified Nucleobases

[0123] Oligomeric compounds may also include nucleobase (often referred to
in the art as heterocyclic base or simply as "base") modifications or
substitutions. As used herein, "unmodified" or "natural" nucleobases
include the purine bases adenine (A) and guanine (G), and the pyrimidine
bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl (--C≡C--CH3) uracil and cytosine and
other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,
8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,
5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted
uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,
2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified
nucleobases include tricyclic pyrimidines such as phenoxazine
cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine
cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such
as a substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazole
cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine
(H-pyrido(3',':4,5)pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases
may also include those in which the purine or pyrimidine base is replaced
with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,
2-aminopyridine and 2-pyridone. Further nucleobases include those
disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise
Encyclopedia Of Polymer Science And Engineering, pages 858-859,
Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by
Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613,
and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. Certain of these nucleobases are particularly useful for
increasing the binding affinity of the compounds of the invention. These
include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6
substituted purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown to
increase nucleic acid duplex stability by 0.6-1.2° C. and are
presently suitable base substitutions, even more particularly when
combined with 2'-O-methoxyethyl sugar modifications.

[0124] Representative U.S. patents that teach the preparation of certain
of the above noted modified nucleobases as well as other modified
nucleobases include, but are not limited to, the above noted U.S. Pat.
No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;
5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;
5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;
5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941,
each of which is herein incorporated by reference, and U.S. Pat. No.
5,750,692, which is herein incorporated by reference.

[0125] Oligomeric compounds of the present invention can also include
polycyclic heterocyclic compounds in place of one or more heterocyclic
base moieties. A number of tricyclic heterocyclic compounds have been
previously reported. These compounds are routinely used in antisense
applications to increase the binding properties of the modified strand to
a target strand. The most studied modifications are targeted to
guanosines hence they have been termed G-clamps or cytidine analogs.
Representative cytosine analogs that make 3 hydrogen bonds with a
guanosine in a second strand include 1,3-diazaphenoxazine-2-one
(R10═O, R11--R14═H) (Kurchavov et al., Nucleosides
and Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one
(R10═S, R11--R14═H), (Lin et al, J. Am. Chem.
Soc., 1995, 117, 3873-3874) and
6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R10═O,
R11--R14═F) (Wang et al, Tetrahedron Lett., 1998, 39,
8385-8388). Incorporated into oligonucleotides these base modifications
were shown to hybridize with complementary guanine and the latter was
also shown to hybridize with adenine and to enhance helical thermal
stability by extended stacking interactions (also see U.S. patent
application entitled "Modified Peptide Nucleic Acids" filed May 24, 2002,
Ser. No. 10/155,920; and U.S. patent application entitled "Nuclease
Resistant Chimeric Oligonucleotides" filed May 24, 2002, Ser. No.
10/013,295, both of which are commonly herein incorporated by reference
in their entirety).

[0126] Further helix-stabilizing properties have been observed when a
cytosine analog/substitute has an aminoethoxy moiety attached to the
rigid 1,3-diazaphenoxazine-2-one scaffold (R10═O,
R11=--O--(CH2)2--NH2, R12-14═H) (Lin et al,
J. Am. Chem. Soc., 1998, 120, 8531-8532). Binding studies demonstrated
that a single incorporation could enhance the binding affinity of a model
oligonucleotide to its complementary target DNA or RNA with a
ΔTm of up to 18° relative to 5-methyl cytosine
(dC5me), which is the highest known affinity enhancement for a
single modification, yet. On the other hand, the gain in helical
stability does not compromise the specificity of the oligonucleotides.

[0127] Further tricyclic heterocyclic compounds and methods of using them
that are amenable to use in the present invention are disclosed in U.S.
Pat. No. 6,028,183, which issued on May 22, 2000, and U.S. Pat. No.
6,007,992, which issued on Dec. 28, 1999, the contents of which are
incorporated herein in their entirety.

[0128] The enhanced binding affinity of the phenoxazine derivatives
together with their uncompromised sequence specificity makes them
valuable nucleobase analogs for the development of more potent
antisense-based drugs. In fact, promising data have been derived from in
vitro experiments demonstrating that heptanucleotides containing
phenoxazine substitutions are capable to activate RNase H, enhance
cellular uptake and exhibit an increased antisense activity (Lin et al,
J. Am. Chem. Soc., 1998, 120, 8531-8532). The activity enhancement was
even more pronounced in case of G-clamp, as a single substitution was
shown to significantly improve the in vitro potency of a 20mer
2'-deoxyphosphorothioate oligonucleotides (Flanagan et al, Proc. Natl.
Acad. Sci. USA, 1999, 96, 3513-3518). Nevertheless, to optimize
oligonucleotide design and to better understand the impact of these
heterocyclic modifications on the biological activity, it is important to
evaluate their effect on the nuclease stability of the oligomers.

[0130] Another modification of the antisense compounds of the invention
involves chemically linking to the oligomeric compound one or more
moieties or conjugates which enhance the activity, cellular distribution
or cellular uptake of the oligonucleotide. These moieties or conjugates
can include conjugate groups covalently bound to functional groups such
as primary or secondary hydroxyl groups. Conjugate groups of the
invention include intercalators, reporter molecules, polyamines,
polyamides, polyethylene glycols, poly-ethers, groups that enhance the
pharmacodynamic properties of oligomers, and groups that enhance the
pharmacokinetic properties of oligomers. Typical conjugate groups include
cholesterols, lipids, phospho-lipids, biotin, phenazine, folate,
phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,
coumarins, and dyes. Groups that enhance the pharmacodynamic properties,
in the context of this invention, include groups that improve uptake,
enhance resistance to degradation, and/or strengthen sequence-specific
hybridization with the target nucleic acid. Groups that enhance the
pharmacokinetic properties, in the context of this invention, include
groups that improve uptake, distribution, metabolism or excretion of the
compounds of the present invention. Representative conjugate groups are
disclosed in International Patent Application PCT/US92/09196, filed Oct.
23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are
incorporated herein by reference. Conjugate moieties include but are not
limited to lipid moieties such as a cholesterol moiety, cholic acid, a
thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,
or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
Antisense compounds of the invention may also be conjugated to active
drug substances, for example, aspirin, warfarin, phenylbutazone,
ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,
carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug
conjugates and their preparation are described in U.S. patent application
Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by
reference in its entirety.

[0132] Oligomeric compounds can also be modified to have one or more
stabilizing groups that are generally attached to one or both termini of
an oligomeric strand to enhance properties such as for example nuclease
stability. Included in stabilizing groups are cap structures. By "cap
structure or terminal cap moiety" is meant chemical modifications, which
have been incorporated at either terminus of oligonucleotides (see for
example Wincott et al., WO 97/26270, incorporated by reference herein.
These terminal modifications protect the oligomeric compounds having
terminal nucleic acid molecules from exonuclease degradation, and can
help in delivery and/or localization within a cell. The cap can be
present at either the 5'-terminus (5'-cap) or at the 3'-terminus (3'-cap)
or can be present on both termini of a single strand, or one or more
termini of both strands of a double-stranded compound. This cap structure
is not to be confused with the inverted methylguanosine "5' cap" present
at the 5' end of native mRNA molecules. In non-limiting examples, the
5'-cap includes inverted abasic residue (moiety), 4',5'-methylene
nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide,
carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;
alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic
3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl riucleotide,
3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety;
3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety;
1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or
bridging or non-bridging methylphosphonate moiety (for more details see
Wincott et al., International PCT publication No. WO 97/26270,
incorporated by reference herein). For siRNA constructs, the 5' end (5'
cap) is commonly but not limited to 5'-hydroxyl or 5'-phosphate.

[0134] Further 3' and 5'-stabilizing groups that can be used to cap one or
both ends of an oligomeric compound to impart nuclease stability include
those disclosed in WO 03/004602 published on Jan. 16, 2003.

Chimeric Compounds

[0135] It is not necessary for all positions in a given antisense compound
to be uniformly modified, and in fact more than one of the aforementioned
modifications may be incorporated in a single compound or even within a
single nucleoside within an antisense compound.

[0136] The present invention also includes antisense compounds which are
chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the
context of this invention, are single- or double-stranded antisense
compounds, such as oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e., a
nucleotide in the case of an oligonucleotide compound. Chimeric antisense
oligonucleotides are one form of antisense compound. These
oligonucleotides typically contain at least one region which is modified
so as to confer upon the oligonucleotide increased resistance to nuclease
degradation, increased cellular uptake, alteration of charge, increased
stability and/or increased binding affinity for the target nucleic acid.
An additional region of the oligonucleotide may serve as a substrate for
RNAses or other enzymes. By way of example, RNAse H is a cellular
endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
Activation of RNase H, therefore, results in cleavage of the RNA target,
thereby greatly enhancing the efficiency of oligonucleotide-mediated
inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in
like fashion, be accomplished through the actions of endoribonucleases,
such as RNase III or RNAseL which cleaves both cellular and viral RNA.
Cleavage products of the RNA target can be routinely detected by gel
electrophoresis and, if necessary, associated nucleic acid hybridization
techniques known in the art.

[0137] Chimeric antisense compounds of the invention may be formed as
composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as
described above. Such compounds have also been referred to in the art as
hybrids or gapmers. Representative United States patents that teach the
preparation of such hybrid structures include, but are not limited to,
U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;
5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and
5,700,922, each of which is herein incorporated by reference in its
entirety.

Salts, Prodrugs and Bioequivalents

[0138] The antisense compounds of the invention encompass any
pharmaceutically acceptable salts, esters, or salts of such esters, or
any other compound which, upon administration to an animal including a
human, is capable of providing (directly or indirectly) the biologically
active metabolite or residue thereof. Accordingly, for example, the
disclosure is also drawn to prodrugs and pharmaceutically acceptable
salts of the compounds of the invention, pharmaceutically acceptable
salts of such prodrugs, and other bioequivalents.

[0139] The term "prodrug" indicates a therapeutic agent that is prepared
in an inactive or less active form that is converted to an active form
(i.e., drug) within the body or cells thereof by the action of endogenous
enzymes or other chemicals and/or conditions. In particular, prodrug
versions of the oligonucleotides of the invention are prepared as SATE
((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods
disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in
WO 94/26764 to Imbach et al.

[0140] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the compounds of
the invention: i.e., salts that retain the desired biological activity of
the parent compound and do not impart undesired toxicological effects
thereto.

[0141] Pharmaceutically acceptable base addition salts are formed with
metals or amines, such as alkali and alkaline earth metals or organic
amines. Examples of metals used as cations are sodium, potassium,
magnesium, calcium, and the like. Examples of suitable amines are
N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,
dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see,
for example, Berge et al., "Pharmaceutical Salts," J. of Pharma Sci.,
1977, 66, 1-19). The base addition salts of said acidic compounds are
prepared by contacting the free acid form with a sufficient amount of the
desired base to produce the salt in the conventional manner. The free
acid form may be regenerated by contacting the salt form with an acid and
isolating the free acid in the conventional manner. The free acid forms
differ from their respective salt forms somewhat in certain physical
properties such as solubility in polar solvents, but otherwise the salts
are equivalent to their respective free acid for purposes of the present
invention. As used herein, a "pharmaceutical addition salt" includes a
pharmaceutically acceptable salt of an acid form of one of the components
of the compositions of the invention. These include organic or inorganic
acid salts of the amines. Acid salts are the hydrochlorides, acetates,
salicylates, nitrates and phosphates. Other suitable pharmaceutically
acceptable salts are well known to those skilled in the art and include
basic salts of a variety of inorganic and organic acids, such as, for
example, with inorganic acids, such as for example hydrochloric acid,
hydrobromic acid, sulfuric acid or phosphoric acid; with organic
carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic
acids, for example acetic acid, propionic acid, glycolic acid, succinic
acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid,
malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid,
glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid,
mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic
acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic
acid; and with amino acids, such as the 20 alpha-amino acids involved in
the synthesis of proteins in nature, for example glutamic acid or
aspartic acid, and also with phenylacetic acid, methanesulfonic acid,
ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic
acid, benzenesulfonic acid, 4-methylbenzenesulfoc acid,
naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or
3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with
the formation of cyclamates), or with other acid organic compounds, such
as ascorbic acid. Pharmaceutically acceptable salts of compounds may also
be prepared with a pharmaceutically acceptable cation. Suitable
pharmaceutically acceptable cations are well known to those skilled in
the art and include alkaline, alkaline earth, ammonium and quaternary
ammonium cations. Carbonates or hydrogen carbonates are also possible.

[0143] The compounds of the invention may also be admixed, encapsulated,
conjugated or otherwise associated with other molecules, molecule
structures or mixtures of compounds, as for example, liposomes,
receptor-targeted molecules, oral, rectal, topical or other formulations,
for assisting in uptake, distribution and/or absorption. Representative
United States patents that teach the preparation of such uptake,
distribution and/or absorption-assisting formulations include, but are
not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.

[0144] The present invention also includes pharmaceutical compositions and
formulations which include the antisense compounds of the invention. The
pharmaceutical compositions of the present invention may be administered
in a number of ways depending upon whether local or systemic treatment is
desired and upon the area to be treated. Administration may be topical
(including ophthalmic and to mucous membranes including vaginal and
rectal delivery), pulmonary, e.g., by inhalation or insufflation of
powders or aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral administration
includes intravenous, intraarterial, subcutaneous, intraperitoneal or
intramuscular injection or infusion; or intracranial, e.g., intrathecal
or intraventricular, administration. Oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly useful for
oral administration. Penetration enhancers have been found to enhance
bioavailability of orally administered oligonucleotides. Penetration
enhancers include surfactants, bile salts, fatty acids, chelating agents
or non-chelating surfactants. Capric acid (C10) and/or lauric acid (C12)
and their salts are among those shown to be effective fatty acids for
enhancing biavailability of oligonucleotides; ursodeoxycholic acid (UDCA)
and chenodeoxycholic acid (CDCA) are among those shown to be effective
bile salts for enhancing biavailability of oligonucleotides.
Delayed-release (for example pulsed or pulsatile-release) formulations
and sustained-release formulations are also useful for enhancing
bioavailability. Bioadhesive materials may be added to adhere drug
carrier particles to mucosal membranes to enhance uptake.

[0145] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily bases,
thickeners and the like may be necessary or desirable. Coated condoms,
gloves and the like may also be useful.

[0146] The pharmaceutical formulations of the present invention, which may
conveniently be presented in unit dosage form, may be prepared according
to conventional techniques well known in the pharmaceutical industry.
Such techniques include the step of bringing into association the active
ingredients with the pharmaceutical carrier(s) or excipient(s). In
general, the formulations are prepared by uniformly and intimately
bringing into association the active ingredients with liquid carriers or
finely divided solid carriers or both, and then, if necessary, shaping
the product.

[0147] The compositions of the present invention may be formulated into
any of many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories, and
enemas. The compositions of the present invention may also be formulated
as suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances which increase the viscosity
of the suspension including, for example, sodium carboxymethylcellulose,
sorbitol and/or dextran. The suspension may also contain stabilizers.

[0148] Pharmaceutical compositions of the present invention include, but
are not limited to, solutions, emulsions, foams and liposome-containing
formulations. The pharmaceutical compositions and formulations of the
present invention may comprise one or more penetration enhancers,
carriers, excipients or other active or inactive ingredients.

[0149] Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1 μm
in diameter. Emulsions may contain additional components in addition to
the dispersed phases, and the active drug which may be present as a
solution in either the aqueous phase, oily phase or itself as a separate
phase. Microemulsions are included as an embodiment of the present
invention. Emulsions and their uses are well known in the art and are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety.

[0150] Formulations of the present invention include liposomal
formulations. As used in the present invention, the term "liposome" means
a vesicle composed of amphiphilic lipids arranged in a spherical bilayer
or bilayers. Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous interior
that contains the composition to be delivered. Cationic liposomes are
positively charged liposomes which are believed to interact with
negatively charged DNA molecules to form a stable complex. Liposomes that
are pH-sensitive or negatively-charged are believed to entrap DNA rather
than complex with it. Both cationic and noncationic liposomes have been
used to deliver DNA to cells.

[0151] Liposomes also include "sterically stabilized" liposomes, a term
which, as used herein, refers to liposomes comprising one or more
specialized lipids that, when incorporated into liposomes, result in
enhanced circulation lifetimes relative to liposomes lacking such
specialized lipids. Examples of sterically stabilized liposomes are those
in which part of the vesicle-forming lipid portion of the liposome
comprises one or more glycolipids or is derivatized with one or more
hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
Liposomes and their uses are further described in U.S. Pat. No.
6,287,860, which is incorporated herein in its entirety.

[0152] The pharmaceutical formulations and compositions of the present
invention may also include surfactants. The use of surfactants in drug
products, formulations and in emulsions is well known in the art.
Surfactants and their uses are further described in U.S. Pat. No.
6,287,860, which is incorporated herein in its entirety.

[0155] Complex formulations containing one or more bile salts and one or
more fatty acids were even more effective, particularly CDCA (with or
without UDCA), in combination with laurate and caprate (U.S. application
Ser. No. 09/108,673, Teng and Hardee, filed Jul. 1, 1998).

[0156] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.

[0158] For topical or other administration, oligonucleotides of the
invention may be encapsulated within liposomes or may form complexes
thereto, in particular to cationic liposomes. Alternatively,
oligonucleotides may be complexed to lipids, in particular to cationic
lipids. Fatty acids and esters, pharmaceutically acceptable salts
thereof, and their uses are further described in U.S. Pat. No. 6,287,860,
which is incorporated herein in its entirety. Topical formulations are
described in detail in U.S. patent application Ser. No. 09/315,298 filed
on May 20, 1999, which is incorporated herein by reference in its
entirety.

[0159] Compositions and formulations for oral administration include
powders or granules, microparticulates, nanoparticulates, suspensions or
solutions in water or non-aqueous media, capsules, gel capsules, sachets,
tablets or minitablets. Thickeners, flavoring agents, diluents,
emulsifiers, dispersing aids or binders may be desirable. Oral
formulations are those in which oligonucleotides of the invention are
administered in conjunction with one or more penetration enhancers
surfactants and chelators. Surfactants include fatty acids and/or esters
or salts thereof, bile acids and/or salts thereof. Bile acids/salts and
fatty acids and their uses are further described in U.S. Pat. No.
6,287,860, which is incorporated herein in its entirety. Also suitable
are combinations of penetration enhancers, for example, fatty acids/salts
in combination with bile acids/salts. One combination is the sodium salt
of lauric acid, capric acid and UDCA. Further penetration enhancers
include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Oligonucleotides of the invention may be delivered orally, in granular
form including sprayed dried particles, or complexed to form micro or
nanoparticles. Oligonucleotide complexing agents and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety. Oral formulations for oligonucleotides and their
preparation are described in detail in U.S. application Ser. Nos.
09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and
10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by
reference in their entirety.

[0160] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous solutions
which may also contain buffers, diluents and other suitable additives
such as, but not limited to, penetration enhancers, carrier compounds and
other pharmaceutically acceptable carriers or excipients.

[0161] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or more
other chemotherapeutic agents which function by a non-antisense
mechanism. Examples of such chemotherapeutic agents include but are not
limited to cancer chemotherapeutic drugs such as daunorubicin,
daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin,
esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,
dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,
mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,
vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,
topotecan, gemcitabine, teniposide, cisplatin, pemetrexed and
diethylstilbestrol (DES). When used with the compounds of the invention,
such chemotherapeutic agents may be used individually (e.g., 5-FU and
oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a
period of time followed by MTX and oligonucleotide), or in combination
with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and
oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).
Anti-inflammatory drugs, including but not limited to nonsteroidal
anti-inflammatory drugs and corticosteroids, and antiviral drugs,
including but not limited to ribivirin, vidarabine, acyclovir and
ganciclovir, may also be combined in compositions of the invention.
Combinations of antisense compounds and other non-antisense drugs are
also within the scope of this invention. Two or more combined compounds
may be used together or sequentially.

[0162] In another related embodiment, compositions of the invention may
contain one or more antisense compounds, particularly oligonucleotides,
targeted to a first nucleic acid and one or more additional antisense
compounds targeted to a second nucleic acid target. Alternatively,
compositions of the invention may contain two or more antisense compounds
targeted to different regions of the same nucleic acid target. Numerous
examples of antisense compounds are known in the art. Two or more
combined compounds may be used together or sequentially.

H. Dosing

[0163] As used herein, the term "patient" refers to a mammal that is
afflicted with one or more disorders associated with eIF4E expression or
overexpression. It will be understood that the most desired patient is a
human. It is also understood that this invention relates specifically to
the inhibition of mammalian eIF4E expression or overexpression.

[0164] It is recognized that one skilled in the art may affect the
disorders associated with eIF4E expression or overexpression by treating
a patient presently afflicted with the disorders with an effective amount
of a compound of the present invention. Thus, the terms "treatment" and
"treating" are intended to refer to all processes wherein there may be a
slowing, interrupting, arresting, controlling, delaying or stopping of
the progression of the disorders described herein, but does not
necessarily indicate a total elimination of all symptoms.

[0165] As used herein, the term "effective amount" or "therapeutically
effective amount" of a compound of the present invention refers to an
amount that is effective in treating or preventing the disorders
described herein.

[0166] The formulation of therapeutic compositions and their subsequent
administration (dosing) is believed to be within the skill of those in
the art. Dosing is dependent on severity and responsiveness of the
disease state to be treated, with the course of treatment lasting from
several days to several months, or until a cure is effected or a
diminution of the disease state is achieved. Optimal dosing schedules can
be calculated from measurements of drug accumulation in the body of the
patient. Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides, and can
generally be estimated based on EC50s found to be effective in in
vitro and in vivo animal models. In general, dosage is from 0.0001 μg
to 100 g per kg of body weight, and may be given once or more daily,
weekly, monthly or yearly, or even once every 2 to 20 years. In some
embodiments, dosage is from 0.0001 μg to 100 g per kg of body weight,
from 0.001 μg to 10 g per kg of body weight, from 0.01 μg to 1 g
per kg of body weight, from 0.1 μg to 100 mg per kg of body weight,
from 1 μg to 10 mg per kg of body weight, from 10 μg to 1 mg per kg
of body weight, or from 100 μg to 500 μg per kg of body weight, and
may be given once or more daily, weekly, monthly or yearly, or even once
every 2 to 20 years. For double-stranded compounds, the dose must be
calculated to account for the increased nucleic acid load of the second
strand (for compounds comprising two strands) or additional nucleic acid
length (for a self-complementary compound). Persons of ordinary skill in
the art can easily estimate repetition rates for dosing based on measured
residence times and concentrations of the drug in bodily fluids or
tissues.

[0167] Much work has been done on the absorbance, distribution, metabolism
and excretion (collectively known as ADME) of oligonucleotides. ADME is
sequence independent because all sequences of a given chemistry (e.g.,
all 2' MOE gapmers with a P═S backbone) have similar
physical/chemical properties such as water solubility, molecular weight
(approx. 7000) and pKa. Oligonucleotides are eliminated relatively
rapidly from plasma (distribution half life approximately 1 hour,
distribution complete by 24 hours) by distribution to tissues, primarily
but not limited to liver, kidney, spleen and bone marrow. A strong
correlation between pharmacokinetics and pharmacodynamics has been
demonstrated in tissues including kidney, liver, bone marrow, adipose
tissue, spleen, lymph nodes, lung (via aerosol) and central nervous
system (given intracerebroventricularly). The tissue half life is 1-5
days for first generation antisense drugs (2'-deoxy with phosphorothioate
backbone) and 10-28 days for 2'-MOE gapped oligonucleotides with
phosphorothioate backbones. Henry et al., Curr. Opin. Invest. Drugs,
2001, 2, 1444-1449.

[0168] Following successful treatment, it may be desirable to have the
patient undergo maintenance therapy to prevent the recurrence of the
disease state, wherein the oligonucleotide is administered in maintenance
doses, ranging from 0.0001 μg to 100 g per kg of body weight, once or
more daily, to once every 20 years.

[0169] While the present invention has been described with specificity in
accordance with certain of its embodiments, the following examples serve
only to illustrate the invention and are not intended to limit the same.
Each of the references, GenBank accession numbers, and the like recited
in the present application is incorporated herein by reference in its
entirety.

[0173] 2'-fluoro oligonucleotides were synthesized as described previously
(Kawasaki et. al., J. Med. Chem., 1993, 36, 831-841) and U.S. Pat. No.
5,670,633, herein incorporated by reference. Briefly, the protected
nucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was synthesized
utilizing commercially available 9-beta-D-arabinofuranosyladenine as
starting material and by modifying literature procedures whereby the
2'-alpha-fluoro atom is introduced by a S.sub.N2-displacement of a
2'-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine
was selectively protected in moderate yield as the
3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and
N6-benzoyl groups was accomplished using standard methodologies and
standard methods were used to obtain the 5'-dimethoxytrityl-(DMT) and
5'-DMT-3'-phosphoramidite intermediates.

[0174] The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished using
tetraisopropyldisiloxanyl (TPDS) protected
9-beta-D-arabinofuranosylguanine as starting material, and conversion to
the intermediate diisobutyrylarabinofuranosylguanosine. Deprotection of
the TPDS group was followed by protection of the hydroxyl group with THP
to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective
O-deacylation and triflation was followed by treatment of the crude
product with fluoride, then deprotection of the THP groups. Standard
methodologies were used to obtain the 5'-DMT- and
5'-DMT-3'-phosphoramidites.

[0175] Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by the
modification of a literature procedure in which
2,2'-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%
hydrogen fluoride-pyridine. Standard procedures were used to obtain the
5'-DMT and 5'-DMT-3' phosphoramidites.

[0176] 2'-deoxy-2'-fluorocytidine was synthesized via amination of
2'-deoxy-2'-fluorouridine, followed by selective protection to give
N4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were used to
obtain the 5'-DMT and 5'-DMT-3' phosphoramidites.

[0178] Aminooxyethyl and dimethylaminooxyethyl amidites are prepared as
per the methods of U.S. Pat. No. 6,127,533 which is herein incorporated
by reference.

Example 2

Oligonucleotide and Oligonucleoside Synthesis

[0179] The oligomeric compounds used in accordance with this invention may
be conveniently and routinely made through the well-known technique of
solid phase synthesis. Equipment for such synthesis is sold by several
vendors including, for example, Applied Biosystems (Foster City, Calif.).
Any other means for such synthesis known in the art may additionally or
alternatively be employed. It is well known to use similar techniques to
prepare oligonucleotides such as the phosphorothioates and alkylated
derivatives. Oligonucleotides: Unsubstituted and substituted
phosphodiester (P═O) oligonucleotides are synthesized on an automated
DNA synthesizer (Applied Biosystems model 394) using standard
phosphoramidite chemistry with oxidation by iodine.

[0180] Phosphorothioates (P═S) are synthesized similar to
phosphodiester oligonucleotides with the following exceptions: thiation
was effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation
of the phosphite linkages. The thiation reaction step time was increased
to 180 sec and preceded by the normal capping step. After cleavage from
the CPG column and deblocking in concentrated ammonium hydroxide at
55° C. (12-16 hr), the oligonucleotides were recovered by
precipitating with >3 volumes of ethanol from a 1 M NH4OAc
solution. Phosphinate oligonucleotides are prepared as described in U.S.
Pat. No. 5,508,270, herein incorporated by reference.

[0181] Alkyl phosphonate oligonucleotides are prepared as described in
U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0182] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as
described in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporated by
reference.

[0183] Phosphoramidite oligonucleotides are prepared as described in U.S.
Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by
reference.

[0184] Alkylphosphonothioate oligonucleotides are prepared as described in
published PCT applications PCT/US94/00902 and PCT/US93/06976 (published
as WO 94/17093 and WO 94/02499, respectively), herein incorporated by
reference.

[0185] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as
described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0186] Phosphotriester oligonucleotides are prepared as described in U.S.
Pat. No. 5,023,243, herein incorporated by reference.

[0187] Borano phosphate oligonucleotides are prepared as described in U.S.
Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.
4'-thio-containing oligonucleotides are synthesized as described in U.S.
Pat. No. 5,639,873, the contents of which are herein incorporated by
reference in their entirety.

[0188] Oligonucleosides: Methylenemethylimino linked oligonucleosides,
also identified as MMI linked oligonucleosides, methylenedimethylhydrazo
linked oligonucleosides, also identified as MDH linked oligonucleosides,
and methylenecarbonylamino linked oligonucleosides, also identified as
amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked oligonucleosides, as
well as mixed backbone compounds having, for instance, alternating MMI
and P═O or P═S linkages are prepared as described in U.S. Pat.
Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of
which are herein incorporated by reference.

[0189] Formacetal and thioformacetal linked oligonucleosides are prepared
as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein
incorporated by reference.

[0190] Ethylene oxide linked oligonucleosides are prepared as described in
U.S. Pat. No. 5,223,618, herein incorporated by reference.

PNA Synthesis

[0191] Peptide nucleic acids (PNAs) are prepared in accordance with any of
the various procedures referred to in Peptide Nucleic Acids (PNA):
Synthesis, Properties and Potential Applications, Bioorganic & Medicinal
Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with
U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated
by reference.

Example 3

RNA Synthesis

[0192] In general, RNA synthesis chemistry is based on the selective
incorporation of various protecting groups at strategic intermediary
reactions. Although one of ordinary skill in the art will understand the
use of protecting groups in organic synthesis, a useful class of
protecting groups includes silyl ethers. In particular bulky silyl ethers
are used to protect the 5'-hydroxyl in combination with an acid-labile
orthoester protecting group on the 2'-hydroxyl. This set of protecting
groups is then used with standard solid-phase synthesis technology. It is
important to lastly remove the acid labile orthoester protecting group
after all other synthetic steps. Moreover, the early use of the silyl
protecting groups during synthesis ensures facile removal when desired,
without undesired deprotection of 2' hydroxyl.

[0193] Following this procedure for the sequential protection of the
5'-hydroxyl in combination with protection of the 2'-hydroxyl by
protecting groups that are differentially removed and are differentially
chemically labile, RNA oligonucleotides were synthesized.

[0194] RNA oligonucleotides are synthesized in a stepwise fashion. Each
nucleotide is added sequentially (3'- to 5'-direction) to a solid
support-bound oligonucleotide. The first nucleoside at the 3'-end of the
chain is covalently attached to a solid support. The nucleotide
precursor, a ribonucleoside phosphoramidite, and activator are added,
coupling the second base onto the 5'-end of the first nucleoside. The
support is washed and any unreacted 5'-hydroxyl groups are capped with
acetic anhydride to yield 5'-acetyl moieties. The linkage is then
oxidized to the more stable and ultimately desired P(V) linkage. At the
end of the nucleotide addition cycle, the 5'-silyl group is cleaved with
fluoride. The cycle is repeated for each subsequent nucleotide.

[0195] Following synthesis, the methyl protecting groups on the phosphates
are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate
(S2Na2) in DMF. The deprotection solution is washed from the
solid support-bound oligonucleotide using water. The support is then
treated with 40% methylamine in water for 10 minutes at 55° C.
This releases the RNA oligonucleotides into solution, deprotects the
exocyclic amines, and modifies the 2'- groups. The oligonucleotides can
be analyzed by anion exchange HPLC at this stage.

[0196] The 2'-orthoester groups are the last protecting groups to be
removed. The ethylene glycol monoacetate orthoester protecting group
developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example
of a useful orthoester protecting group which, has the following
important properties. It is stable to the conditions of nucleoside
phosphoramidite synthesis and oligonucleotide synthesis. However, after
oligonucleotide synthesis the oligonucleotide is treated with methylamine
which not only cleaves the oligonucleotide from the solid support but
also removes the acetyl groups from the orthoesters. The resulting
2-ethyl-hydroxyl substituents on the orthoester are less electron
withdrawing than the acetylated precursor. As a result, the modified
orthoester becomes more labile to acid-catalyzed hydrolysis.
Specifically, the rate of cleavage is approximately 10 times faster after
the acetyl groups are removed. Therefore, this orthoester possesses
sufficient stability in order to be compatible with oligonucleotide
synthesis and yet, when subsequently modified, permits deprotection to be
carried out under relatively mild aqueous conditions compatible with the
final RNA oligonucleotide product.

[0198] RNA antisense compounds (RNA oligonucleotides) of the present
invention can be synthesized by the methods herein or purchased from
Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized,
complementary RNA antisense compounds can then be annealed by methods
known in the art to form double stranded (duplexed) antisense compounds.
For example, duplexes can be formed by combining 30 μl of each of the
complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide
solution) and 15 μl of 5× annealing buffer (100 mM potassium
acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by
heating for 1 minute at 90° C., then 1 hour at 37° C. The
resulting duplexed antisense compounds can be used in kits, assays,
screens, or other methods to investigate the role of a target nucleic
acid, or for diagnostic or therapeutic purposes.

Example 4

Synthesis of Chimeric Oligonucleotides

[0199] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of several
different types. These include a first type wherein the "gap" segment of
linked nucleosides is positioned between 5' and 3' "wing" segments of
linked nucleosides and a second "open end" type wherein the "gap" segment
is located at either the 3' or the 5' terminus of the oligomeric
compound. Oligonucleotides of the first type are also known in the art as
"gapmers" or gapped oligonucleotides. Oligonucleotides of the second type
are also known in the art as "hemimers" or "wingmers".

[0200] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and
2'-deoxy phosphorothioate oligonucleotide segments are synthesized using
an Applied Biosystems automated DNA synthesizer Model 394, as above.
Oligonucleotides are synthesized using the automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and
5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for the 2'-O-alkyl
portion. The standard synthesis cycle is modified by incorporating
coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully protected
oligonucleotide is cleaved from the support and deprotected in
concentrated ammonia (NH4OH) for 12-16 hr at 55° C. The
deprotected oligo is then recovered by an appropriate method
(precipitation, column chromatography, volume reduced in vacuo and
analyzed spetrophotometrically for yield and for purity by capillary
electrophoresis and by mass spectrometry.

[0201] (2'-O-(2-methoxyethyl))--(2'-deoxy)-(-2'-O-(methoxyethyl)) chimeric
phosphorothioate oligonucleotides were prepared as per the procedure
above for the 2'-O-methyl chimeric oligonucleotide, with the substitution
of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites.

[0202] (2'-O-(2-methoxyethyl phosphodiester)-(2'-deoxy
phosphorothioate)-(2'-O-(methoxyethyl) phosphodiester) chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidation with
iodine to generate the phosphodiester internucleotide linkages within the
wing portions of the chimeric structures and sulfurization utilizing
3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate
the phosphorothioate internucleotide linkages for the center gap.

[0203] Other chimeric oligonucleotides, chimeric oligonucleosides and
mixed chimeric oligonucleotides/oligonucleosides are synthesized
according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

Design and Screening of Duplexed Antisense Compounds Targeting eIF4E

[0204] In accordance with the present invention, a series of nucleic acid
duplexes comprising the antisense compounds of the present invention and
their complements can be designed to target eIF4E. The nucleobase
sequence of the antisense strand of the duplex comprises at least an
8-nucleobase portion of an oligonucleotide in Table 1. The ends of the
strands may be modified by the addition of one or more natural or
modified nucleobases to form an overhang. The sense strand of the dsRNA
is then designed and synthesized as the complement of the antisense
strand and may also contain modifications or additions to either
terminus. For example, in one embodiment, both strands of the dsRNA
duplex would be complementary over the central nucleobases, each having
overhangs at one or both termini. It is possible for one end of a duplex
to be blunt and the other to have overhanging nucleobases. In one
embodiment, the number of overhanging nucleobases is from 1 to 6 on the
3' end of each strand of the duplex. In another embodiment, the number of
overhanging nucleobases is from 1 to 6 on the 3' end of only one strand
of the duplex. In a further embodiment, the number of overhanging
nucleobases is from 1 to 6 on one or both 5' ends of the duplexed
strands. In another embodiment, the number of overhanging nucleobases is
zero.

[0205] By way of example, a duplex comprising an antisense strand having
the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:456) and having a
two-nucleobase overhang of deoxythymidine(dT) would have the following
structure:

##STR00001##

[0206] In another embodiment, a duplex comprising an antisense strand
having the same sequence CGAGAGGCGGACGGGACCG may be prepared with blunt
ends (no single stranded overhang) as shown:

##STR00002##

The duplex may be unimolecular or bimolecular, i.e., the sense and
antisense strands may be part of the same molecule (which forms a hairpin
or other self structure) or two (or even more) separate molecules.

[0207] RNA strands of the duplex can be synthesized by methods disclosed
herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).
Once synthesized, the complementary strands are annealed. The single
strands are aliquoted and diluted to a concentration of 50 uM. Once
diluted, 30 uL of each strand is combined with 15 uL of a 5×
solution of annealing buffer. The final concentration of said buffer is
100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium
acetate. The final volume is 75 uL. This solution is incubated for 1
minute at 90° C. and then centrifuged for 15 seconds. The tube is
allowed to sit for 1 hour at 37° C. at which time the dsRNA
duplexes are used in experimentation. The final concentration of the
dsRNA duplex is 20 uM. This solution can be stored frozen (-20°
C.) and freeze-thawed up to 5 times.

[0208] Once prepared, the duplexed antisense compounds are evaluated for
their ability to modulate eIF4E expression.

[0209] When cells reached 80% confluency, they are treated with duplexed
antisense compounds of the invention. For cells grown in 96-well plates,
wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium
(Gibco BRL) and then treated with 130 μL, of OPTI-MEM-1 containing 12
μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound
at a final concentration of 200 nM. After 5 hours of treatment, the
medium is replaced with fresh medium. Cells are harvested 16 hours after
treatment, at which time RNA is isolated and target reduction measured by
RT-PCR.

Example 6

Oligonucleotide Isolation

[0210] After cleavage from the controlled pore glass solid support and
deblocking in concentrated ammonium hydroxide at 55° C. for 12-16
hours, the oligonucleotides or oligonucleosides are recovered by
precipitation out of 1 M NH4OAc with >3 volumes of ethanol.
Synthesized oligonucleotides were analyzed by electrospray mass
spectroscopy (molecular weight determination) and by capillary gel
electrophoresis and judged to be at least 70% full length material. The
relative amounts of phosphorothioate and phosphodiester linkages obtained
in the synthesis was determined by the ratio of correct molecular weight
relative to the -16 amu product (+/-32 +/-48). For some studies
oligonucleotides were purified by HPLC, as described by Chiang et al., J.
Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified material.

[0212] Oligonucleotides were cleaved from support and deprotected with
concentrated NH4OH at elevated temperature (55-60° C.) for
12-16 hours and the released product then dried in vacuo. The dried
product was then re-suspended in sterile water to afford a master plate
from which all analytical and test plate samples are then diluted
utilizing robotic pipettors.

Example 8

Oligonucleotide Analysis--96-Well Plate Format

[0213] The concentration of oligonucleotide in each well was assessed by
dilution of samples and UV absorption spectroscopy. The full-length
integrity of the individual products was evaluated by capillary
electrophoresis (CE) in either the 96-well format (Beckman P/ACE® MDQ)
or, for individually prepared samples, on a commercial CE apparatus
(e.g., Beckman P/ACE® 5000, ABI 270). Base and backbone composition
was confirmed by mass analysis of the compounds utilizing
electrospray-mass spectroscopy. All assay test plates were diluted from
the master plate using single and multi-channel robotic pipettors. Plates
were judged to be acceptable if at least 85% of the compounds on the
plate were at least 85% full length.

[0214] The effect of antisense compounds on target nucleic acid expression
can be tested in any of a variety of cell types provided that the target
nucleic acid is present at measurable levels. This can be routinely
determined using, for example, PCR or Northern blot analysis. The
following cell types are provided for illustrative purposes, but other
cell types can be routinely used, provided that the target is expressed
in the cell type chosen. This can be readily determined by methods
routine in the art, for example Northern blot analysis, ribonuclease
protection assays, or RT-PCR.

[0218] Human neonatal dermal fibroblast (NHDF) were obtained from the
Clonetics Corporation (Walkersville, Md.). NHDFs were routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville, Md.) supplemented as recommended by the supplier. Cells
were maintained for up to 10 passages as recommended by the supplier.

HEK Cells:

[0219] Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained
in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)
formulated as recommended by the supplier. Cells were routinely
maintained for up to 10 passages as recommended by the supplier.

b.END Cells:

[0220] The mouse brain endothelial cell line b.END was obtained from Dr.
Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.END
cells were routinely cultured in DMEM, high glucose (Gibco/Life
Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum
(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely
passaged by trypsinization and dilution when they reached 90% confluence.
Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a
density of 3000 cells/well for use in RT-PCR analysis. For Northern
blotting or other analyses, cells may be seeded onto 100 mm or other
standard tissue culture plates and treated similarly, using appropriate
volumes of medium and oligonucleotide.

HeLa Cells:

[0221] The human epitheloid carcinoma cell line HeLa was obtained from the
American Tissue Type Culture Collection (Manassas, Va.). HeLa cells were
routinely cultured in DMEM, high glucose (Invitrogen Corporation,
Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen
Corporation, Carlsbad, Calif.). Cells were routinely passaged by
trypsinization and dilution when they reached approximately 90%
confluence. Cells were seeded into 24-well plates (Falcon-Primaria #3846)
at a density of approximately 50,000 cells/well or in 96-well plates at a
density of approximately 5,000 cells/well for use in RT-PCR analysis. For
Northern blotting or other analyses, cells were harvested when they
reached approximately 90% confluence.

U-87 MG Cells:

[0222] The human glioblastoma U-87 MG cell line was obtained from the
American Type Culture Collection (Manassas, Va.). U-87 MG cells were
cultured in DMEM (Invitrogen Life Technologies, Carlsbad, Calif.)
supplemented with 10% fetal bovine serum (Invitrogen Life Technologies,
Carlsbad, Calif.) and antibiotics. Cells were routinely passaged by
trypsinization and dilution when they reached appropriate confluence.
Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a
density of about 10,000 cells/well for use in RT-PCR analysis.

[0223] For Northern blotting or other analyses, cells may be seeded onto
100 mm or other standard tissue culture plates and treated similarly,
using appropriate volumes of medium and oligonucleotide. For Northern
blotting or other analyses, cells may be seeded onto 100 mm or other
standard tissue culture plates and treated similarly, using appropriate
volumes of medium and oligonucleotide.

[0225] When cells reached 65-75% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were washed
once with 100 μL OPTI-MEM®-1 reduced-serum medium (Invitrogen
Corporation, Carlsbad, Calif.) and then treated with 130 μL of
OPTI-MEM®-1 containing 3.75 mg/mL LIPOFECTIN® (Invitrogen
Corporation, Carlsbad, Calif.) and the desired concentration of
oligonucleotide. Cells are treated and data are obtained in triplicate.
After 4-7 hours of treatment at 37° C., the medium was replaced
with fresh medium. Cells were harvested 16-24 hours after oligonucleotide
treatment.

[0226] The concentration of oligonucleotide used varies from cell line to
cell line. To determine the optimal oligonucleotide concentration for a
particular cell line, the cells are treated with a positive control
oligonucleotide at a range of concentrations. For human cells the
positive control oligonucleotide is selected from either ISIS 13920
(TCCGTCATCGCTCCTCAGGG, SEQ ID NO:1) which is targeted to human H-ras, or
ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO:2) which is targeted to
human Jun-N-terminal kinase-2 (JNK2). Both controls are 2'-O-methoxyethyl
gapmers (2'-O-methoxyethyls shown in bold) with a phosphorothioate
backbone. For mouse or rat cells the positive control oligonucleotide is
ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO:3, a 2'-O-methoxyethyl gapmer
(2'-O-methoxyethyls shown in bold) with a phosphorothioate backbone which
is targeted to both mouse and rat c-raf. The concentration of positive
control oligonucleotide that results in 80% inhibition of c-H-ras (for
ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then
utilized as the screening concentration for new oligonucleotides in
subsequent experiments for that cell line. If 80% inhibition is not
achieved, the lowest concentration of positive control oligonucleotide
that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then
utilized as the oligonucleotide screening concentration in subsequent
experiments for that cell line. If 60% inhibition is not achieved, that
particular cell line is deemed as unsuitable for oligonucleotide
transfection experiments. The concentrations of antisense
oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

Analysis of Oligonucleotide Inhibition of eIF4E Expression

[0227] Antisense modulation of eIF4E expression can be assayed in a
variety of ways known in the art. For example, eIF4E mRNA levels can be
quantitated by, e.g., Northern blot analysis, competitive polymerase
chain reaction (PCR), or real-time PCR(RT-PCR). Real-time quantitative
PCR is presently suitable. RNA analysis can be performed on total
cellular RNA or poly(A)+ mRNA. One method of RNA analysis of the present
invention is the use of total cellular RNA as described in other examples
herein. Methods of RNA isolation are well known in the art. Northern blot
analysis is also routine in the art. Real-time quantitative (PCR) can be
conveniently accomplished using the commercially available ABI PRISM®
7600, 7700, or 7900 Sequence Detection System, available from PE-Applied
Biosystems, Foster City, Calif. and used according to manufacturer's
instructions.

[0228] Protein levels of eIF4E can be quantitated in a variety of ways
well known in the art, such as immunoprecipitation, Western blot analysis
(immunoblotting), enzyme-linked immunosorbent assay (ELISA) or
fluorescence-activated cell sorting (FACS). Antibodies directed to eIF4E
can be identified and obtained from a variety of sources, such as the
MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can
be prepared via conventional monoclonal or polyclonal antibody generation
methods well known in the art.

Example 11

Design of Phenotypic Assays for the Use of eIF4E Inhibitors

[0229] Once eIF4E inhibitors have been identified by the methods disclosed
herein, the compounds are further investigated in one or more phenotypic
assays, each having measurable endpoints predictive of efficacy in the
treatment of a particular disease state or condition.

[0230] Phenotypic assays, kits and reagents for their use are well known
to those skilled in the art and are herein used to investigate the role
and/or association of eIF4E in health and disease. Representative
phenotypic assays, which can be purchased from any one of several
commercial vendors, include those for determining cell viability,
cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,
Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including
enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin
Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell
regulation, signal transduction, inflammation, oxidative processes and
apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride
accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube
formation assays, cytokine and hormone assays and metabolic assays
(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,
Piscataway, N.J.).

[0231] In one non-limiting example, cells determined to be appropriate for
a particular phenotypic assay (i.e., MCF-7 cells selected for breast
cancer studies; adipocytes for obesity studies) are treated with eIF4E
inhibitors identified from the in vitro studies as well as control
compounds at optimal concentrations which are determined by the methods
described above. At the end of the treatment period, treated and
untreated cells are analyzed by one or more methods specific for the
assay to determine phenotypic outcomes and endpoints.

[0232] Phenotypic endpoints include changes in cell morphology over time
or treatment dose as well as changes in levels of cellular components
such as proteins, lipids, nucleic acids, hormones, saccharides or metals.
Measurements of cellular status which include pH, stage of the cell
cycle, intake or excretion of biological indicators by the cell, are also
endpoints of interest.

[0233] Analysis of the genotype of the cell (measurement of the expression
of one or more of the genes of the cell) after treatment is also used as
an indicator of the efficacy or potency of the eIF4E inhibitors. Hallmark
genes, or those genes suspected to be associated with a specific disease
state, condition, or phenotype, are measured in both treated and
untreated cells.

Example 12

RNA Isolation

[0234] Poly(A)+ mRNA Isolation

[0235] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,
1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are
routine in the art. Briefly, for cells grown on 96-well plates, growth
medium was removed from the cells and each well was washed with 200 μL
cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M
NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each
well, the plate was gently agitated and then incubated at room
temperature for five minutes. 55 μL of lysate was transferred to Oligo
d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were
incubated for 60 minutes at room temperature, washed 3 times with 200
μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to remove
excess wash buffer and then air-dried for 5 minutes. 60 μL of elution
buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to
each well, the plate was incubated on a 90° C. hot plate for 5
minutes, and the eluate was then transferred to a fresh 96-well plate.

[0236] Cells grown on 100 mm or other standard plates may be treated
similarly, using appropriate volumes of all solutions.

Total RNA Isolation

[0237] Total RNA was isolated using an RNEASY96® kit and buffers
purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each well
was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to
each well and the plate vigorously agitated for 20 seconds. 150 μL of
70% ethanol was then added to each well and the contents mixed by
pipetting three times up and down. The samples were then transferred to
the RNEASY96® well plate attached to a QIAVAC® manifold fitted with
a waste collection tray and attached to a vacuum source. Vacuum was
applied for 1 minute. 500 μL of Buffer RW1 was added to each well of
the RNEASY96® plate and incubated for 15 minutes and the vacuum was
again applied for 1 minute. An additional 500 μL of Buffer RW1 was
added to each well of the RNEASY 96® plate and the vacuum was applied
for 2 minutes. 1 mL of Buffer RPE was then added to each well of the
RNEASY96® plate and the vacuum applied for a period of 90 seconds. The
Buffer RPE wash was then repeated and the vacuum was applied for an
additional 3 minutes. The plate was then removed from the QIAVAC®
manifold and blotted dry on paper towels. The plate was then re-attached
to the QIAVAC® manifold fitted with a collection tube rack containing
1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of
RNAse free water into each well, incubating 1 minute, and then applying
the vacuum for 3 minutes.

[0238] The repetitive pipetting and elution steps may be automated using a
QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after
lysing of the cells on the culture plate, the plate is transferred to the
robot deck where the pipetting, DNase treatment and elution steps are
carried out.

Example 13

Real-Time Quantitative PCR Analysis of eIF4E mRNA Levels

[0239] Quantitation of eIF4E mRNA levels was accomplished by real-time
quantitative PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence
Detection System (PE-Applied Biosystems, Foster City, Calif.) according
to manufacturer's instructions. This is a closed-tube, non-gel-based,
fluorescence detection system which allows high-throughput quantitation
of polymerase chain reaction (PCR) products in real-time. As opposed to
standard PCR in which amplification products are quantitated after the
PCR is completed, products in real-time quantitative PCR are quantitated
as they accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the forward
and reverse PCR primers, and contains two fluorescent dyes. A reporter
dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster
City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 5' end of the
probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied
Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda,
Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached
to the 3' end of the probe. When the probe and dyes are intact, reporter
dye emission is quenched by the proximity of the 3' quencher dye. During
amplification, annealing of the probe to the target sequence creates a
substrate that can be cleaved by the 5'-exonuclease activity of Taq
polymerase. During the extension phase of the PCR amplification cycle,
cleavage of the probe by Taq polymerase releases the reporter dye from
the remainder of the probe (and hence from the quencher moiety) and a
sequence-specific fluorescent signal is generated. With each cycle,
additional reporter dye molecules are cleaved from their respective
probes, and the fluorescence intensity is monitored at regular intervals
by laser optics built into the ABI PRISM® Sequence Detection System.
In each assay, a series of parallel reactions containing serial dilutions
of mRNA from untreated control samples generates a standard curve that is
used to quantitate the percent inhibition after antisense oligonucleotide
treatment of test samples.

[0240] Prior to quantitative PCR analysis, primer-probe sets specific to
the target gene being measured are evaluated for their ability to be
"multiplexed" with a GAPDH amplification reaction. In multiplexing, both
the target gene and the internal standard gene GAPDH are amplified
concurrently in a single sample. In this analysis, mRNA isolated from
untreated cells is serially diluted. Each dilution is amplified in the
presence of primer-probe sets specific for GAPDH only, target gene only
("single-plexing"), or both (multiplexing). Following PCR amplification,
standard curves of GAPDH and target mRNA signal as a function of dilution
are generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of their
corresponding values generated from the single-plexed samples, the
primer-probe set specific for that target is deemed multiplexable. Other
methods of PCR are also known in the art.

[0242] Gene target quantities obtained by real time RT-PCR are normalized
using either the expression level of GAPDH, a gene whose expression is
constant, or by quantifying total RNA using RiboGreen® (Molecular
Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time
RT-PCR, by being run simultaneously with the target, multiplexing, or
separately. Total RNA is quantified using RiboGreen® RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of
RNA quantification by RiboGreen® are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374).

[0244] Probes and primers to human eIF4E were designed to hybridize to a
human eIF4E sequence, using published sequence information (GenBank
accession number M15353.1, incorporated herein as SEQ ID NO: 4). For
human eIF4E the PCR primers were:

[0245] forward primer: TGGCGACTGTCGAACCG (SEQ ID NO:5)

[0246] reverse primer: AGATTCCGTTTTCTCCTCTTCTGTAG (SEQ ID NO:6)

[0247] and the PCR probe was: FAM-AAACCACCCCTACTCCTAATCCCCCG-TAMRA (SEQ ID
NO:7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For
human GAPDH the PCR primers were:

[0248] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)

[0249] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9)

[0250] and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID
NO:10) where JOE is the fluorescent reporter dye and TAMRA is the
quencher dye.

[0251] Probes and primers to mouse eIF4E were designed to hybridize to a
mouse eIF4E sequence, using published sequence information (GenBank
accession number NM--007917.1, incorporated herein as SEQ ID NO:11).
For mouse eIF4E the PCR primers were:

[0252] forward primer: AGGACGGTGGCTGATCACA (SEQ ID NO:12)

[0253] reverse primer: TCTCTAGCCAGAAGCGATCGA (SEQ ID NO:13)

[0254] and the PCR probe was: FAM-TGAACAAGCAGCAGAGACGGAGTGA-TAMRA (SEQ ID
NO:14) where FAM is the fluorescent reporter dye and TAMRA is the
quencher dye. For mouse GAPDH the PCR primers were:

[0255] forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO:15)

[0256] reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:16)

[0257] and the PCR probe was: 5' JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3'
(SEQ ID NO:17) where JOE is the fluorescent reporter dye and TAMRA is the
quencher dye.

[0262] In accordance with the present invention, a series of antisense
compounds were designed to target different regions of the human eIF4E
RNA, using published sequences (GenBank accession number M15353.1,
incorporated herein as SEQ ID NO:4). The compounds are shown in Table 1.
"Target site" indicates the first (5'-most) nucleotide number on the
particular human eIF4E target sequence to which the compound binds. All
compounds in Table 1 are chimeric oligonucleotides ("gapmers") 20
nucleotides in length, composed of a central "gap" region consisting of
ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3'
directions) by five-nucleotide "wings". The wings are composed of
2'-methoxyethyl (2'-MOE) nucleotides. The internucleoside (backbone)
linkages are phosphorothioate (P═S) throughout the oligonucleotide.
All cytidine residues are 5-methylcytidines.

[0263] A second series of antisense compounds were designed to target
different regions of the mouse eIF4E RNA, using published sequences
(GenBank accession number NM--007917.1, incorporated herein as SEQ
ID NO:11). The compounds are shown in Table 1. "Target site" indicates
the first (5'-most) nucleotide number on the particular human eIF4E
target nucleic acid to which the compound binds. All compounds in Table 1
are chimeric oligonucleotides ("gapmers") 20 nucleotides in length,
composed of a central "gap" region consisting of ten 2'-deoxynucleotides,
which is flanked on both sides (5' and 3' directions) by five-nucleotide
"wings". The wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides.
The internucleoside (backbone) linkages are phosphorothioate (P═S)
throughout the oligonucleotide. All cytidine residues are
5-methylcytidines.

[0264] As compounds in Table 1 are complementary to both human and mouse
eIF4E sequences, the compounds were analyzed for their effect on human
eIF4E mRNA levels by quantitative real-time PCR as described in other
examples herein. Data are averages from three experiments in which A549
cells were treated with the antisense oligonucleotides of the present
invention. The positive control for each datapoint is identified in the
table by sequence ID number. If present, "N.D." indicates "no data".

[0266] The target regions to which these suitable sequences are
complementary are herein referred to as "suitable target segments" and
are therefore suitable for targeting by compounds of the present
invention. These suitable target segments are shown in Table 3. These
sequences are shown to contain thymine (T) but one of skill in the art
will appreciate that thymine (T) is generally replaced by uracil (U) in
RNA sequences. The sequences represent the reverse complement of the
suitable antisense compounds shown in Table 1. "Target site" indicates
the first (5'-most) nucleotide number on the particular target nucleic
acid to which the oligonucleotide binds. Also shown in Table 3 is the
species in which each of the suitable target segments was found.

[0267] In accordance with the present invention, the compounds in Table 1,
which are complementary to both human and mouse eIF4E (for example mouse
eIF4E GenBank accession number NM--007917.1, incorporated herein as
SEQ ID NO:11) were further analyzed for their effect on mouse eIF4E mRNA
levels by quantitative real-time PCR as described in other examples
herein. In Table 2, "target site" indicates the first (5'-most)
nucleotide number on the particular mouse eIF4E target nucleic acid to
which the compound binds. Data, shown in Table 2, are averages from three
experiments in which b.END cells were treated with the antisense
oligonucleotides of the present invention. The positive control for each
datapoint is identified in the table by sequence ID number. If present,
"N.D." indicates "no data".

[0269] The target regions to which these suitable sequences are
complementary are herein referred to as "suitable target segments" and
are therefore suitable for targeting by compounds of the present
invention. These suitable target segments are shown in Table 3. These
sequences are shown to contain thymine (T) but one of skill in the art
will appreciate that thymine (T) is generally replaced by uracil (U) in
RNA sequences. The sequences represent the reverse complement of the
suitable antisense compounds shown in Tables 1 and 2. "Target site"
indicates the first (5'-most) nucleotide number on the particular target
nucleic acid to which the oligonucleotide binds. Also shown in Table 3 is
the species in which each of the suitable target segments was found.

[0270] As these "suitable target segments" have been found by
experimentation to be open to, and accessible for, hybridization with the
antisense compounds of the present invention, one of skill in the art
will recognize or be able to ascertain, using no more than routine
experimentation, further embodiments of the invention that encompass
other compounds that specifically hybridize to these suitable target
segments and consequently inhibit the expression of eIF4E.

[0271] According to the present invention, antisense compounds include
antisense oligonucleotides, ribozymes, external guide sequence (EGS)
oligonucleotides, siRNA compounds, single- or double-stranded RNA
interference (RNAi) compounds and other oligomeric compounds which
hybridize to at least a portion of the target nucleic acid and modulate
its function.

Example 17

Western Blot Analysis of eIF4E Protein Levels

[0272] Western blot analysis (immunoblot analysis) may be carried out
using standard methods. Cells are harvested 16-20 h after oligonucleotide
treatment, washed once with PBS, suspended in Laemmli buffer (100
μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels
are run for 1.5 hours at 150 V, and transferred to membrane for western
blotting. Appropriate primary antibody directed to eIF4E is used, with a
radiolabeled or fluorescently labeled secondary antibody directed against
the primary antibody species. Bands are visualized using a
PHOSPHORIMAGER® (Molecular Dynamics, Sunnyvale Calif.).

Example 18

Effect of Antisense Inhibition of eIF4E Expression on Cell Proliferation

[0274] eIF4A target mRNA reduction was also measured in this experiment.
Both ISIS 183750 and ISIS 299815 yielded IC50s of less than 3 μM
(concentration needed to inhibit eIF4E mRNA levels by 50%) and showed
70-80% inhibition at oligonucleotide concentrations of 7.5 μM and
higher. Control oligonucleotides 29848 and 129688 yielded a maximum
inhibition of 20% (7.5 μM 129688) but generally gave approximately 10%
inhibition at other concentrations.

[0275] The effect of antisense inhibition of eIF4E on cell proliferation
was also measured in U87-MG human glioblastoma cells. U87-MG cells
(American Type Culture Collection, Manassas Va.), 1×106
cells/100 μl, were electroporated with ISIS 183750 (SEQ ID NO:40) and
ISIS 298815 (SEQ ID NO:97) and an unrelated (control) oligonucleotide
ISIS 129699 (GGATAGAACGCGAAAGCTTG; SEQ ID NO:209) at 7.5 μM. The two
antisense inhibitors of eIF4E, ISIS 183750 and ISIS 298815, reduced
U87-MG cell proliferation compared to control (ISIS 129699) by
approximately 12% and 10%, respectively, after 96 hours. EIF4E target
mRNA was measured at 48 hours after start of treatment and was reduced by
approximately 31% by ISIS 183750 and 36% by ISIS 298815 when compared to
untreated control. eIF4E mRNA levels were not reduced by control
oligonucleotide ISIS 129699 and actually increased slightly.

Example 19

Effect of Antisense Inhibition of eIF4E Expression on Cell Cycle

[0276] The effect of eIF4E antisense compounds on the cell cycle was
examined. HeLa cells were electroporated with 30 μM antisense
oligonucleotide (ISIS 183750 or 299815) or control oligonucleotide (ISIS
29848 or ISIS 129688), or mock transfected. The fluorescent DNA
intercalator propidium iodide (PI) was used to measure DNA content at 48
hours, using flow cytometry. Results (done in duplicate) are shown in
Table 4.

[0277] From the results shown in Table 4 it can be seen that treatment
with both eIF4E antisense compounds (ISIS 183750 or ISIS 298815)
increased the portion of cells in SubG1 phase, which is generally
indicative of apoptosis. The portion of cells in G2M are also increased
after ISIS 298815 treatment.

Example 20

Effect of Antisense Inhibition of eIF4E Expression on Angiogenesis/Tube
Formation

[0278] Angiogenesis is stimulated by numerous factors that promote
interaction of endothelial cells with each other and with extracellular
matrix molecules, resulting in the formation of capillary tubes. This
process can be reproduced in tissue culture by the formation of tube-like
structures by endothelial cells. Loss of tube formation in vitro has been
correlated with the inhibition of angiogenesis in vivo (Carmeliet et al.,
Nature, 2000, 407, 249-257; and Zhang et al., Cancer Research, 2002, 62,
2034-42), which supports the use of in vitro tube formation as an
endpoint for angiogenesis.

[0279] The tube formation assay is performed using an In vitro
Angiogenesis Assay Kit (Chemicon International, Temecula, Calif.), or
growth factor reduced Matrigel (BD Biosciences, Bedford, Mass.). HUVECs
were plated at 4000 cells/well in 96-well plates. One day later, cells
were transfected with antisense and control oligonucleotides according to
standard published procedures (Monia et al., J. Biol. Chem., 1993,
268(19), 14514-22) using 75 nM oligonucleotide in lipofectin (Gibco,
Grand Island, N.Y.). Approximately fifty hours post-transfection, cells
were transferred to 96-well plates coated with ECMatrix® (Chemicon
Inter-national) or growth factor depleted Matrigel. Under these
conditions, untreated HUVECs form tube-like structures. After an
overnight incubation at 37° C., treated and untreated cells were
inspected by light microscopy. Individual wells were assigned discrete
scores from 1 to 5 depending on the extent of tube formation. A score of
1 refers to a well with no tube formation while a score of 5 is given to
wells where all cells are forming an extensive tubular network.

[0280] As calculated from the assigned discrete scores, cells treated with
antisense inhibitors ISIS 183750 and ISIS 298815 had average tube
formation scores of approximately 1.5 and 2.25, respectively. Cells
treated with the random control oligonucleotide ISIS 29848
(NNNNNNNNNNNNNNNNNNNN; SEQ ID NO:207, wherein N is a mixture of A, C, G
and T) had an average tube formation score of approximately 4.25 and
cells treated with ISIS 334163 (TGTTACAGTCTTGTACCCTT; SEQ ID NO:210), a
6-base mismatch of ISIS 183750, had an average tube formation score of
approximately 4.5. Thus, tube formation is specifically inhibited by
47-67% by eIF4E antisense oligonucleotides. Antisense inhibitors of eIF4E
can, therefore, inhibit angiogenesis.

Example 21

Inhibition of eIF4E Expression in Mice

[0281] Eight-week old C57BL6 mice were injected intraperitoneally with
oligonucleotide in saline twice weekly for 3 weeks (6 doses total) at an
oligonucleotide concentration of 40 mg/kg. Compounds used were eIF4E
antisense compounds ISIS 183750 (SEQ ID NO:40), ISIS 299815 (SEQ ID
NO:97), ISIS 298797 (SEQ ID NO:80) and ISIS 298823 (SEQ ID NO:105). All
are cross-species antisense oligonucleotides to both human and mouse
eIF4E. ISIS 141923 is an unrelated (control) oligonucleotide
(CCTTCCCTGAAGGTTCCTCC; SEQ ID NO:211). A saline (vehicle) control was
also used. Compared to saline control, ISIS 183750 reduced eIF4E mRNA
levels in mouse liver to less than 20% of control (over 80% inhibition).
ISIS 298815 also reduced eIF4E mRNA levels to approximately 20% of
control. ISIS 298797 treatment reduced eIF4E mRNA levels to approximately
30% of control (70% inhibition) and ISIS 298823 treatment reduced eIF4E
mRNA levels to approximately 37% of control (63% inhibition). In
contrast, treatment with ISIS 141923 did not reduce eIF4E mRNA levels and
actually increased them to approximately 140% of saline control.

[0283] Mice treated with any one of the eIF4E antisense compounds showed
essentially no changes in liver, spleen or total body weight. There was
no significant change in liver enzyme levels (AST/ALT) and liver
histology appeared the same as for saline-treated control mice.

Example 22

Effect of Antisense Inhibition of eIF4E Expression on Human Tumor
Xenografts in Mice

[0284] Male nude mice were injected subcutaneously in the flank with
5×106 PC-3 human prostate carcinoma cells (American Type
Culture Collection, Manassas Va.). Antisense treatment began when the
tumors reached a mean size of 100 mm3, approximately 3 to 3.5 weeks
after implantation. Mice were given 50 mg/kg by intravenous injection of
antisense to eIF4E, ISIS 183750 (SEQ ID NO:40) or control oligonucleotide
ISIS 141923 (SEQ ID NO:211) on the first dose and then 25 mg/kg every
Monday, Wednesday and Friday thereafter. By day 54 after tumor
implantation, tumors in mice treated with ISIS 183750 were approximately
450 mm3 in size, approximately a 50% reduction compared to tumors in
mice treated with control, ISIS 141923 (approximately 930 mm3. This
level of reduction continued until the end of study at day 57.

[0285] Xenografts were also done similarly using MDA-231 human breast
cancer cells (American Type Culture Collection, Manassas Va.) in female
mice. In this experiment both ISIS 183750 and ISIS 298815 were tested and
gave nearly identical reduction in tumor cell growth of 55% and 50%,
respectively, compared to saline control. eIF4E protein expression was
measured in these MDA-231 xenografts by Western blot analysis (using
antibody to eIF4E from Pharmingen, San Diego Calif.) and was found to be
reduced by 45% in mice treated with ISIS 183750 (SEQ ID NO:40) and by 39%
in mice treated with ISIS 298815 (SEQ ID NO:97), when compared to
xenografts in mice treated with an unrelated control oligonucleotide
(ISIS 141923, SEQ ID NO:211).

[0286] eIF4E can be phosphorylated in vivo at serine residue 209 of the
human sequence. The phosphorylated form is often regarded as the active
state of the protein, with increased phosphorylation often correlated
with upregulation of rates of protein synthesis. Western blots using
antibody specific for phosphorylated (pS209) eIF4E (BioSource,
Camarillo Calif.) confirmed a decrease in the phosphorylated form of
eIF4E after treatment with antisense compounds ISIS 183750 and 298815,
but not an antisense control (ISIS 129699).

[0287] Cyclin D1 is an eIF4E target protein and cyclin D1 protein was also
found to be reduced in MDA-231 xenografts in mice treated with antisense
to eIF4E. Cyclin D1 was reduced by 40% after treatment with ISIS 183750
and by nearly 50% after treatment with ISIS 298815, when compared to
cyclin D1 expression in xenografts in mice treated with unrelated control
oligonucleotide ISIS 141923.

[0288] In a third similarly conducted xenograft study, female nude mice
were injected subcutaneously into the flank with 5×106 H460
human non-small-cell lung cancer (NSCLC) cells (American Type Culture
Collection, Manassas Va.). Intravenous dosing with oligonucleotides began
once the tumors reached a mean size of 100 mm3. The antisense
treatment schedule began with a single dose of ISIS 141923 or ISIS 183750
at 50 mg/kg followed thereafter by 25 mg/kg every Monday, Wednesday and
Friday for a total treatment time of 17 days. At the end of the study,
the mean tumor volume of the ISIS 141923 control-treated group was
approximately 2000 mm3 vs. 550 mm3 for ISIS 183750
(p<0.001).

Example 23

Inhibition of eIF-4E by Short Double Stranded RNA Oligonucleotides Design
and Synthesis of dsRNA Oligonucleotides

[0289] Human eIF-4E sequence Genbank #M15353 was queried for sequences.
The G+C content of selected sequences range from 30% to 70%. Each of the
dsRNA sequences specific to eIF-4E and depicted below contain two
deoxythymidine nucleotides at the 3' terminal end of each strand of the
RNA oligonucleotide duplex (not shown). Synthesis, duplex formation and
purification of gene-specific siRNAs was performed by Dharmacon Research
Inc. Three eIF-4E siRNA sequences were selected and tested, and are shown
below:

[0296] A control dsRNA compound, also containing two deoxythymidine
nucleotides at the 3' terminal end of each strand and complementary to
pGL3 Luciferase, was purchased from Dharmacon Research Inc. and used in
the assays below.

[0299] Mammalian cell lines are plated at 1×105 cells in
24-well plates, 24 hours prior to transfection. Transient transfections
are performed using Oligofectamine (Invitrogen). Briefly, individual
dsRNAs at a concentration between 5 to 500 nM (final volume) are diluted
in OptiMEM (Invitrogen) while a separate solution of OptiMEM and
Oligofectamine is incubated at room temperature for 5 min. The two
solutions are mixed, followed by a 30-minute room temperature incubation.
Serum-containing media is added to the transfection complex for a final
volume of 0.5 ml/well. Existing cell media is aspirated and replaced with
the transfection complex and incubated for 48-72 h at 37° C., 5%
CO2.

[0301] In the LNCaP cell line, each of eIF4E-1, eIF4E-2, and eIF4E-3
inhibited eIF-4E protein levels by greater than 50% at concentrations of
less than 50 nM. In the CWR22RV1 cell line, concentrations of less than 5
nM eIF4E-2 inhibited eIF-4E protein levels by greater than 50%. In each
of the LNCaP, PC3, HCT116, MDA-231, and MCF-7 cell lines, concentrations
of eIF4E-1 and eIF4E-2 less than 50 nM reduced eIF-4E protein expression
by greater than 50%.

Cell Proliferation Assays

[0302] Cells are plated 24 hours prior to transfection at a cell density
between 1.5-3.0×103 cells/well in poly-D-lysine coated 96-well
plates (Becton Dickinson). Transfections of the eIF-4E and control siRNAs
are performed in triplicate at siRNA concentrations ranging from 5 nM to
500 nM. Cells are harvested at 3, 6 and 8 days by addition of propidium
iodide (Sigma) at 50 μg/ml final concentration, followed by a 30
minute room temperature incubation protected from light. Plates are
measured pre- and post-freezing on a Victor2 1420 multi-label
counter (Wallac). Corrected sums are obtained by subtracting the pre-from
the post-freeze measurements.

[0303] In each of the LNCaP, PC3, and MDA-231 cell lines, concentrations
of eIF4E-1 and eIF4E-2 less than 50 nM reduced cell proliferation by
greater than 50%.

Example 24

Activity of siRNA Constructs Targeted to eIF4E in HeLa Cells

[0304] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 5
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. For comparison several single stranded
chimeric antisense oligonucleotides were also tested.

[0305] Cells were plated in 96-well plates at a density of 5000 cells/well
and grown in DMEM with high glucose, 10% FBS, 1% penicillin/streptomycin.
Wells were washed once with 200 μL OPTI-MEM-1® reduced-serum medium
(Gibco BRL) and then treated with 130 μL of OPTI-MEM-1® containing
the desired dsRNA at a concentration of 25 nM and 2.5 ul/ml
LIPOFECTIN® (Gibco BRL) per strand of oligomeric compound. Treatments
were done in duplicate. After 4 or 5 hours of treatment, the medium was
replaced with fresh medium. Cells were harvested 16 or 18 hours after
dsRNA treatment, at which time RNA was isolated and target reduction
measured by RT-PCR as described in previous examples, normalized to
Ribogreen. Human primer/probe set is SEQ ID NO:5, 6 and 7 used in
previous examples.

[0306] The results are shown in Table 5. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 5 below, followed by the sense strand
(S) in the next row. Unless otherwise indicated, all double-stranded
constructs are unmodified RNA, i.e., ribose sugars with phosphate
(P═O) backbones and 5'-terminal hydroxyl group, and are blunt-ended
(no dTdT or other overhang) unless otherwise indicated. Unless otherwise
indicated, single-stranded antisense molecules are chimeric gapped
oligonucleotides with 2'-MOE at nucleotides 1-5 and 16-20 and
2'-deoxynucleotides at positions 6-15, with phosphorothioate (P═S)
backbones and 5-methylcytosines at every C. It is understood in the art
that, for RNA sequences, U (uracil) generally replaces T (thymine) which
is normally found in DNA or DNA-like sequences.

[0307] In this screen, the MOE gapmer leads to eIF4E were found to be
slightly more active (94% inhibition) than the best siRNA (88%
inhibition). Three out of five siRNA constructs at previously identified
MOE gapmer lead sites are active. Eight eIF4E siRNA constructs show
target reduction of 70% or more, and seven show reduction of 75% or more.
This is consistent with the conclusions of Vickers et al. (J. Biol.
Chem., 2003, 278, 7108-7118), i.e., in general, activity of siRNA
oligonucleotide duplexes correlated with the activity of RNase
H-dependent oligonucleotides (e.g, MOE gapmers) targeted to the same
site, and optimized siRNA and RNase H-dependent oligonucleotides behave
similarly in terms of potency, maximal effects, specificity and duration
of action and efficiency.

[0308] The compounds in the above table were also tested for the ability
to reduce PTEN RNA levels in HeLa cells. None of the eIF4E-targeted
compounds (siRNA or single stranded MOE gapmers) reduced PTEN target RNA
levels by more than about 20%. The siRNA positive control 335449
construct inhibited PTEN RNA by about 85% and the single stranded MOE
gapmer positive control ISIS 116847 inhibited PTEN RNA by about 80%.

Example 25

Activity of siRNA Constructs Targeted to eIF4E in MH-S Cells

[0309] Nearly all of the siRNA compounds in the previous table are
perfectly complementary to both mouse and human eIF4E mRNA. Here they are
tested in the mouse MH-S murine alveolar macrophage cell line. Mouse MH-S
cells were purchased from the American Type Culture Collection (Manassas,
Va.). The cells were maintained in RPMI 1640 medium containing 10%
heat-inactivated fetal calf serum (FCS) (Hyclone Laboratories, Logan,
Utah). Cells were plated in 96-well plates at a density of 5000
cells/well and grown in DMEM with high glucose, 10% FBS, 1%
penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a concentration
of 20 nM and 2.5 ul/ml LIPOFECTIN® (Gibco BRL) per strand of
oligomeric compound. Treatments were done in duplicate. After 4 or 5
hours of treatment, the medium was replaced with fresh medium. Cells were
harvested 16 or 18 hours after dsRNA treatment, at which time RNA was
isolated and target reduction measured by RT-PCR as described in previous
examples.

[0310] The results are shown in Table 6. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 6 below, followed by the sense strand
(S) in the next row. Unless otherwise indicated, all double-stranded
constructs are unmodified RNA, i.e., ribose sugars with phosphate
(P═O) backbones and 5'-terminal hydroxyl group. Unless otherwise
indicated, single-stranded antisense molecules are chimeric gapped
oligonucleotides with 2'-MOE at nucleotides 1-5 and 16-20 and
2'-deoxynucleotides at positions 6-15, with phosphorothioate (P═S)
backbones and 5-methylcytosines at every C. It is understood in the art
that, for RNA sequences, U (uracil) generally replaces T (thymine) which
is normally found in DNA or DNA-like sequences.

[0311] Target sites, species, chemistry and sequences are as in previous
tables.

[0312] An additional gene walk was done to identify additional siRNAs that
inhibit eIF4E. Constructs were screened in HeLa cells at a concentration
of 50 nM.

[0313] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 7
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. For comparison several single stranded
chimeric antisense oligonucleotides were also tested.

[0314] Cells were plated in 96-well plates at a density of 5000 cells/well
and grown in DMEM with high glucose, 10% FBS, 1% penicillin/streptomycin.
Wells were washed once with 200 μL OPTI-MEM-1® reduced-serum medium
(Gibco BRL) and then treated with 130 μL of OPTI-MEM-1® containing
the desired dsRNA at a concentration of 50 nM and 2.5 ul/ml
LIPOFECTIN® (Gibco BRL) per strand of oligomeric compound. Treatments
were done in duplicate. After 4 or 5 hours of treatment, the medium was
replaced with fresh medium. Cells were harvested 16 or 18 hours after
dsRNA treatment, at which time RNA was isolated and target reduction
measured by RT-PCR as described in previous examples.

The results are shown in Table 7. The siRNA constructs shown consist of
one antisense strand and one sense strand. The antisense strand (AS) is
shown first in Table 7 below, followed by the sense strand (S) in the
next row. Unless otherwise indicated, all double-stranded constructs are
unmodified RNA, i.e., ribose sugars with phosphate (P═O) backbones
and 5'-terminal hydroxyl group. Unless otherwise indicated,
single-stranded antisense molecules are chimeric gapped oligonucleotides
with 2'-MOE at nucleotides 1-5 and 16-20 and 2'-deoxynucleotides at
positions 6-15, with phosphorothioate (P═S) backbones and
5-methylcytosines at every C. It is understood in the art that, for RNA
sequences, U (uracil) generally replaces T (thymine) which is normally
found in DNA or DNA-like sequences.

[0315] Nearly all of the siRNA compounds in the previous table are
perfectly complementary to both mouse and human eIF4E mRNA. Here they are
tested in the mouse MH-S murine alveolar macrophage cell line. Mouse MH-S
cells were purchased from the American Type Culture Collection (Manassas,
Va.). The cells were maintained in RPMI 1640 medium containing 10%
heat-inactivated fetal calf serum (FCS) (Hyclone Laboratories, Logan,
Utah). Cells were plated in 96-well plates at a density of 5000
cells/well and grown in DMEM with high glucose, 10% FBS, 1%
penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a concentration
of 50 nM and 2.5 ul/ml LIPOFECTIN® (Gibco BRL) per strand of
oligomeric compound. Treatments were done in duplicate. After 4 or 5
hours of treatment, the medium was replaced with fresh medium. Cells were
harvested 16 or 18 hours after dsRNA treatment, at which time RNA was
isolated and target reduction measured by RT-PCR as described in previous
examples. The results are shown in Table 8. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown; sense strand, target site, species, chemistry and
sequence are as in previous tables. It is understood in the art that, for
RNA sequences, U (uracil) generally replaces T (thymine) which is
normally found in DNA or DNA-like sequences.

[0317] A dose-response experiment was done in HeLa cells using above
treatment methods and siRNA concentrations of 0.1 nM, 1.0 nM, 10 nM and
100 nM, and an IC50 (concentration of compound resulting in 50%
inhibition of eIF4E compared to untreated control) was calculated for
certain of the above compounds. The results are shown in Table 9.
Antisense strand identity is shown. Sense strand, target site, species,
chemistry and sequence are as in previous tables.

Four of the above siRNA constructs were chosen for further evaluation and
SAR (structure-activity-relationship) analysis. These parent constructs
for siRNA SAR analysis are as shown here. It is understood in the art
that, for RNA sequences, U (uracil) generally replaces T (thymine) which
is normally found in DNA or DNA-like sequences.

[0318] The four siRNA constructs chosen in the previous example ("parent"
constructs) were compared to siRNA constructs that have alternating
2'-O-methyl (2'-O-Me or 2'OMe) and 2'-fluoro (2'-F) modifications.

[0319] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 10
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. For comparison several single stranded
chimeric antisense oligonucleotides were also tested. Cells were plated
in 96-well plates at a density of 5000 cells/well and grown in DMEM with
high glucose, 10% FBS, 1% penicillin/streptomycin. Wells were washed once
with 200 μL OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then
treated with 130 μL of OPTI-MEM-1® containing the desired dsRNA at
concentrations of 0.2, 2 and 20 nM and 2.5 μl/ml LIPOFECTIN® (Gibco
BRL) per strand of oligomeric compound. Treatments were done in
duplicate. After 4 or 5 hours of treatment, the medium was replaced with
fresh medium. Cells were harvested 16 or 18 hours after dsRNA treatment,
at which time RNA was isolated and target reduction measured by RT-PCR as
described in previous examples.

[0320] The results are shown in Table 10. The siRNA constructs shown
consist of one antisense strand and one sense strand. For the alternating
2'-OMe/2'-F modified compounds, both the sense and antisense strands were
modified, with the 5'-most nucleoside on the sense strand being a 2'-F
and the 5'-most nucleoside on the antisense strand being a 2'-O-Me, so
that the two kinds of modification are out of register in the duplexed
molecule. It should be noted that the parent compounds are 20mers and the
2' modified compounds shown are 19mers, lacking the base pair
corresponding to the 5' most pair of the sense strand (i.e., of the
duplex as shown) These are shown in Table 10. 2'-O-methyl nucleosides are
shown in bold; 2'-fluoro are underlined. Unmodified ribose is shown in
plain UPPERCASE text. It is understood in the art that, for RNA
sequences, U (uracil) generally replaces T (thymine) which is normally
found in DNA or DNA-like sequences.

[0321] For several of the constructs, the alternating
2'-O-methyl/2'-fluoro (2'-OMe/2'F) construct was shown to be comparable
to or better than the parent (unmodified RNA) construct in efficacy of
eIF4E mRNA reduction. Furthermore, the stability of the modified
construct tested was more than 8-fold that of the unmodified compound
(details in following example).

[0322] Intact duplex RNA was analyzed from diluted mouse-plasma using an
extraction and capillary electrophoresis method similar to those
previously described (Leeds et al., Anal. Biochem., 1996, 235, 36-43;
Geary et al., Anal. Biochem., 1999, 274, 241-248). Heparin-treated mouse
plasma, from 3-6 month old female Balb/c mice (Charles River Labs) was
thawed from -80° C. and diluted to 25% (v/v) with phosphate
buffered saline (140 mM NaCl, 3 mM KCl, 2 mM potassium phosphate, 10 mM
sodium phosphate). Approximately 10 nmol of pre-annealed siRNA, at a
concentration of 100 μM, was added to the 25% plasma and incubated at
37° C. for 0, 15, 30, 45, 60, 120, 180, 240, 360, and 420 minutes.
Aliquots were removed at the indicated time, treated with EDTA to a final
concentration of 2 mM, and placed on ice at 0° C. until analyzed
by capillary gel electrophoresis (Beckman P/ACE MDQ-UV with eCap DNA
Capillary tube). The area of the siRNA duplex peak was measured and used
to calculate the percent of intact siRNA remaining. Adenosine
triphosphate (ATP) was added at a concentration of 2.5 mM to each
injection as an internal calibration standard. A zero time point was
taken by diluting siRNA in phosphate buffered saline followed by
capillary electrophoresis. Percent intact siRNA was plotted against time,
allowing the calculation of a pseudo first-order half-life. Results are
shown in Table 11.

[0323] The parent (unmodified) construct is approximately 50% degraded
after 30 minutes and nearly gone after 4 hours (completely gone at 6
hours). In contrast, the alternating 2'-O-methyl/2'-fluoro construct
remains relatively unchanged and 75% remains even after 6 hours.

Example 31

Additional Modifications of eIF4E siRNA

[0324] Additional siRNA constructs with various modifications were
prepared and tested as described in previous examples.

[0325] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 12
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. Cells were plated in 96-well plates at a
density of 5000 cells/well and grown in DMEM with high glucose, 10% FBS,
1% penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a range of
concentrations and 2.5 μl/ml LIPOFECTIN® (Gibco BRL) per strand of
oligomeric compound. Treatments were done in duplicate. After 4 or 5
hours of treatment, the medium was replaced with fresh medium. Cells were
harvested 16 or 18 hours after dsRNA treatment, at which time RNA was
isolated and target reduction measured by RT-PCR as described in previous
examples. For stability analysis, siRNA duplexes were incubated in 25%
heparinized mouse plasma at 37° C. and analyzed by capillary gel
electrophoresis with an internal reference standard.

[0326] The results are shown in Table 12. The siRNA constructs shown
consist of one antisense strand and one sense strand. Unless otherwise
indicated, all double-stranded constructs are unmodified RNA, i.e.,
ribose sugars with phosphate (P═O) backbones and 5'-terminal hydroxyl
group. Unless otherwise indicated, single-stranded antisense molecules
are chimeric gapped oligonucleotides with 2'-MOE at nucleotides 1-5 and
16-20 and 2'-deoxynucleotides at positions 6-15, with phosphorothioate
(P═S) backbones and 5-methylcytosines at every C. It is understood in
the art that, for RNA sequences, U (uracil) generally replaces T
(thymine) which is normally found in DNA or DNA-like sequences.
2'-O-methyl nucleosides are shown in bold; 2'-fluoro are underlined,
4'-thio nucleosides are shown in lower case and unmodified ribose is
shown in plain UPPERCASE text.

[0328] 345847--345849 is a 19mer with alternating ribose and 2'OMe
nucleosides (out of register) on both strands. The 5' most nucleoside of
the sense strand is ribose and the 5' most nucleoside of the antisense
strand is 2'OMe.

[0329] 351831--351832 is a 19mer with alternating 2'-F and 2'OMe
nucleosides (out of register) on both strands. The 5' most nucleoside of
the sense strand is 2'-F and the 5' most nucleoside of the antisense
strand is 2' OMe.

[0330] 352824--342764 is a 19mer with three 4'-thio nucleosides at
each terminus of the antisense strand (sense strand is unmodified).

[0331] 352827--342764 is a 19mer with three 4'-thio nucleosides at
the 5' terminus of the antisense strand and three 2'-OMe nucleosides at
the 3' terminus of the antisense strand (sense strand is unmodified).

[0332] 349890--338935 is a 20mer with mixed 2'-F/2'-OMe modifications
of the antisense strand (sense strand is unmodified). The antisense
strand has 2'-F at positions 1-5, 8, 9, and 12-17 and 2'-OMe at positions
6, 7, 10, 11, 18-20 (starting at the 5' end).

[0333] 349891--338939 is a 20mer with mixed 2'-F/2'-OMe modifications
of the antisense strand (sense strand is unmodified). The antisense
strand has 2'-F at positions 1-5, 8, 9, and 12-17 and 2'-OMe at positions
6, 7, 10, 11, 18-20 (starting at the 5' end).

[0334] 351097--338952 is a 20mer with mixed 2'-F/2'-OMe modifications
of the antisense strand (sense strand is unmodified). The antisense
strand has 2'-F at positions 1-5, 8, 9, and 12-17 and 2'-OMe at positions
6, 7, 10, 11, 18-20 (starting at the 5' end).

[0335] It should be noted that the parent compounds are 20mers and some of
the 2' modified compounds shown are 19mers, lacking the base pair
corresponding to the 5' most pair of the sense strand (i.e., of the
duplex as shown) These are shown in Table 12. 2'-O-methyl nucleosides are
shown in bold; 2'-fluoro are underlined, 4'-thio nucleosides are shown in
lower case and unmodified ribose is shown in plain UPPERCASE text.

[0336] The mixed (block) 2'-O-methyl/2'-fluoro (2'-OMe/2'F) construct
349892--338943 was shown to be comparable to or better than the
parent (unmodified RNA) construct in efficacy of eIF4E mRNA reduction.

[0337] The alternating 2'-O-methyl/unmodified construct
345847--345849 construct was tested twice and was also shown to be
comparable to or better than the parent (unmodified RNA) construct in
efficacy of eIF4E mRNA reduction, with enhanced stability.

[0338] The 4'-thio block modified construct 352824--342764 was less
active than the parent but highly stable.

[0339] The 4'-thio/2'-O-methyl construct 352827--342764 was
comparable to the parent in efficacy. Stability data has not yet been
obtained.

Example 32

Gapped Modified siRNA Constructs--Activity in HeLa Cells

[0340] Additional siRNA constructs were tested in HeLa cells. The duplexed
oligomeric RNA (dsRNA) compounds shown in Table 13 below were prepared as
described in previous examples and evaluated in HeLa cells (American Type
Culture Collection, Manassas Va.). Culture methods used for HeLa cells
are available from the ATCC and may be found, for example, at
www.atcc.org. Cells were plated in 96-well plates at a density of 5000
cells/well and grown in DMEM with high glucose, 10% FBS, 1%
penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at concentrations of
0.1, 1, 10 and 100 nM and 2.5 μl/ml LIPOFECTIN® (Gibco BRL) per
strand of oligomeric compound. Treatments were done in duplicate. After 4
or 5 hours of treatment, the medium was replaced with fresh medium. Cells
were harvested 16 or 18 hours after dsRNA treatment, at which time RNA
was isolated and target reduction measured by RT-PCR as described in
previous examples.

[0341] For stability analysis, siRNA duplexes were incubated in 25%
heparinized mouse plasma at 37° C. and analyzed by capillary gel
electrophoresis with an internal reference standard. The results are
shown in Table 13. The siRNA constructs shown consist of one antisense
strand and one sense strand. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group. Unless
otherwise indicated, single-stranded antisense molecules are chimeric
gapped oligonucleotides with 2'-MOE at nucleotides 1-5 and 16-20 and
2'-deoxynucleotides at positions 6-15, with phosphorothioate (P═S)
backbones and 5-methylcytosines at every C. It is understood in the art
that, for RNA sequences, U (uracil) generally replaces T (thymine) which
is normally found in DNA or DNA-like sequences.

[0343] 349896--338943 has 2'F at positions 1-5, ribose at positions
6-15, and 2'Ome at positions 16-20 of the antisense strand, counting from
the 5' end of the AS strand; the sense strand is unmodified (ribose,
P═O backbone).

[0344] 349894--338935 has 2'F at positions 1-5, ribose at positions
6-15, and 2'Ome at positions 16-20 of the antisense strand, counting from
the 5' end of the AS strand; the sense strand is unmodified (ribose,
P═O backbone).

[0345] 349895--338939 has 2'F at positions 1-5, ribose at positions
6-15, and 2'Ome at positions 16-20 of the antisense strand, counting from
the 5' end of the AS strand; the sense strand is unmodified (ribose,
P═O backbone).

[0346] 349897--338952 has 2'F at positions 1-5, ribose at positions
6-15, and 2'Ome at positions 16-20 of the antisense strand, counting from
the 5' end of the AS strand; the sense strand is unmodified (ribose,
P═O backbone).

[0347] These are shown in Table 13. 2'-O-methyl nucleosides are shown in
bold; 2'-fluoro are underlined, 4'-thio nucleosides are shown in lower
case and unmodified ribose is shown in plain text. It is understood in
the art that, for RNA sequences, U (uracil) generally replaces T
(thymine) which is normally found in DNA or DNA-like sequences.

[0348] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 14
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Cells were
plated in 96-well plates at a density of 5000 cells/well and grown in
DMEM with high glucose, 10% FBS, 1% penicillin/streptomycin. Wells were
washed once with 200 μL OPTI-MEM-1® reduced-serum medium (Gibco
BRL) and then treated with 130 μL of OPTI-MEM-1® containing the
desired dsRNA at a concentration of 0.2, 2 and 20 nM plus 2.5 μl/ml
LIPOFECTIN® (Gibco BRL) per strand of oligomeric compound. Treatments
were done in duplicate. After 4 or 5 hours of treatment, the medium was
replaced with fresh medium. Cells were harvested 16 or 18 hours after
dsRNA treatment, at which time RNA was isolated and target reduction
measured by RT-PCR as described in previous examples.

[0349] The results are shown in Table 14. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 14 below, followed by the sense
strand (S) in the next row. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group. It is
understood in the art that, for RNA sequences, U (uracil) generally
replaces T (thymine) which is normally found in DNA or DNA-like
sequences. 2'-O-methyl nucleosides are shown in bold.

[0350] 338932 is an unmodified ribose 20mer with phosphate (P═O)
backbone and 5' phosphate, targeted to the site of the eIF4E--1
(341887) 19mer.

[0364] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 15
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. Cells were plated in 96-well plates at a
density of 5000 cells/well and grown in DMEM with high glucose, 10% FBS,
1% penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a concentration
of 0.2, 2 and 20 nM plus 2.5 μl/ml LIPOFECTIN® (Gibco BRL) per
strand of oligomeric compound. Treatments were done in duplicate. After 4
or 5 hours of treatment, the medium was replaced with fresh medium. Cells
were harvested 16 or 18 hours after dsRNA treatment, at which time RNA
was isolated and target reduction measured by RT-PCR as described in
previous examples.

[0365] The results are shown in Table 15. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 15 below, followed by the sense
strand (S) in the next row. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group. It is
understood in the art that, for RNA sequences, U (uracil) generally
replaces T (thymine) which is normally found in DNA or DNA-like
sequences. 2'-O-methyl nucleosides are shown in bold.

[0366] 338932 is an unmodified ribose 20mer with phosphate (P═O)
backbone and 5' terminal phosphate, targeted to the eIF4E--1 site.

[0376] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 16
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. Cells were plated in 96-well plates at a
density of 5000 cells/well and grown in DMEM with high glucose, 10% FBS,
1% penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a concentration
of 0.02, 0.2, 2 and 20 nM with 2.5 μl/ml LIPOFECTIN® (Gibco BRL)
per strand of oligomeric compound. Treatments were done in duplicate.
After 4 or 5 hours of treatment, the medium was replaced with fresh
medium. Cells were harvested 16 or 18 hours after dsRNA treatment, at
which time RNA was isolated and target reduction measured by RT-PCR as
described in previous examples.

[0377] The results are shown in Table 16. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 16 below, followed by the sense
strand (S) in the next row. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group. It is
understood in the art that, for RNA sequences, U (uracil) generally
replaces T (thymine) which is normally found in DNA or DNA-like
sequences. 2'-O-methyl nucleosides are shown in bold, unmodified ribose
nucleosides are in PLAIN UPPERCASE and 4' thio are in lower case. All
sequences in Table 16 are 19mers of SEQ ID NO: 301 (antisense strand)/SEQ
ID NO: 302 (sense strand).

[0386] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 17
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. Cells were plated in 96-well plates at a
density of 5000 cells/well and grown in DMEM with high glucose, 10% FBS,
1% penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a concentration
of 0.5, 5 and 50 nM with 2.5 μl/ml LIPOFECTIN® (Gibco BRL) per
strand of oligomeric compound. Treatments were done in duplicate. After 4
or 5 hours of treatment, the medium was replaced with fresh medium. Cells
were harvested 16 or 18 hours after dsRNA treatment, at which time RNA
was isolated and target reduction measured by RT-PCR as described in
previous examples.

[0387] The results are shown in Table 17. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 17 below, followed by the sense
strand (S) in the next row. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group. It is
understood in the art that, for RNA sequences, U (uracil) generally
replaces T (thymine) which is normally found in DNA or DNA-like
sequences. 2'-O-methyl nucleosides are shown in bold, unmodified ribose
nucleosides are in PLAIN UPPERCASE.

[0395] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 18
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. Cells were plated in 96-well plates at a
density of 5000 cells/well and grown in DMEM with high glucose, 10% FBS,
1% penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a concentration
0.5, 5 and 50 nM with 2.5 μl/ml LIPOFECTIN® (Gibco BRL) per strand
of oligomeric compound. Treatments were done in duplicate. After 4 or 5
hours of treatment, the medium was replaced with fresh medium. Cells were
harvested 16 or 18 hours after dsRNA treatment, at which time RNA was
isolated and target reduction measured by RT-PCR as described in previous
examples.

[0396] The results are shown in Table 18. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 18 below, followed by the sense
strand (S) in the next row. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group. It is
understood in the art that, for RNA sequences, U (uracil) generally
replaces T (thymine) which is normally found in DNA or DNA-like
sequences. 2'-O-methyl nucleosides are shown in bold.

[0405] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 19
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. Cells were plated in 96-well plates at a
density of 5000 cells/well and grown in DMEM with high glucose, 10% FBS,
1% penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a concentration
0.5, 5 and 50 nM with 2.5 μl/ml LIPOFECTIN® (Gibco BRL) per strand
of oligomeric compound. Treatments were done in duplicate. After 4 or 5
hours of treatment, the medium was replaced with fresh medium. Cells were
harvested 16 or 18 hours after dsRNA treatment, at which time RNA was
isolated and target reduction measured by RT-PCR as described in previous
examples.

[0406] The results are shown in Table 19. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 19 below, followed by the sense
strand (S) in the next row. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group It is
understood in the art that, for RNA sequences, U (uracil) generally
replaces T (thymine) which is normally found in DNA or DNA-like
sequences. 2'-O-methyl nucleosides are shown in bold.

[0415] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 20
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. Cells were plated in 96-well plates at a
density of 5000 cells/well and grown in DMEM with high glucose, 10% FBS,
1% penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a concentration
0.5, 5 and 50 nM with 2.5 μl/ml LIPOFECTIN® (Gibco BRL) per strand
of oligomeric compound. Treatments were done in duplicate. After 4 or 5
hours of treatment, the medium was replaced with fresh medium. Cells were
harvested 16 or 18 hours after dsRNA treatment, at which time RNA was
isolated and target reduction measured by RT-PCR as described in previous
examples.

[0416] The results are shown in Table 20. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 20 below, followed by the sense
strand (S) in the next row. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group. It is
understood in the art that, for RNA sequences, U (uracil) generally
replaces T (thymine) which is normally found in DNA or DNA-like
sequences. 2'-O-methyl nucleosides are shown in bold.

[0424] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 21
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Culture
methods used for HeLa cells are available from the ATCC and may be found,
for example, at www.atcc.org. Cells were plated in 96-well plates at a
density of 5000 cells/well and grown in DMEM with high glucose, 10% FBS,
1% penicillin/streptomycin. Wells were washed once with 200 μL
OPTI-MEM-1® reduced-serum medium (Gibco BRL) and then treated with 130
μL of OPTI-MEM-1® containing the desired dsRNA at a concentration
of 0.02, 0.2, 2 and 20 nM with 2.5 μl/ml LIPOFECTIN® (Gibco BRL)
per strand of oligomeric compound. Treatments were done in duplicate.
After 4 or 5 hours of treatment, the medium was replaced with fresh
medium. Cells were harvested 16 or 18 hours after dsRNA treatment, at
which time RNA was isolated and target reduction measured by RT-PCR as
described in previous examples.

[0425] The results are shown in Table 21. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 21 below, followed by the sense
strand (S) in the next row. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group. It is
understood in the art that, for RNA sequences, U (uracil) generally
replaces T (thymine) which is normally found in DNA or DNA-like
sequences. 2'-O-methyl nucleosides are shown in bold. All are 19mers of
SEQ ID NO: 301 (antisense strand)/SEQ ID NO: 302 (sense strand).

[0434] The modified or unmodified siRNA constructs shown in previous
tables are tested for their ability to reduce levels of human eIF4E mRNA
in U-87 MG cells using the methods described above. The U-87 human
glioblastoma cell line is obtained from the ATCC (Rockville Md.) and
maintained in Iscove's DMEM medium supplemented with heat-inactivated 10%
fetal calf serum. Dose response experiments are performed as described in
previous examples to obtain IC50 values.

Example 42

Additional siRNAs Targeted to Human eIF4E

[0435] Additional siRNAs were designed to human eIF4E mRNA (Genbank
accession no. M15353.1, SEQ ID NO: 4) All are alternating
2'-O-methyl/2'-OH on antisense strand and alternating 2'-OH/2'-O-methyl
on sense strand. The backbone is phosphate (P═O) and the 5' terminus
is 5'-OH, although it will be understood that these and other siRNA
constructs shownherein may also be synthesized with a 5'-phosphate group.

[0436] The antisense strands are shown in Table 22; sense strands are
fully complementary and are not shown.

[0437] "Target site" refers to the 5'-most position of the target region
on the M15353.1 human eIF4E sequence (SEQ ID NO: 4) to which the
oligonucleotide is targeted.

[0438] A set of uniform 2'-O-methoxyethyl (2'-MOE) phosphorothioate
oligonucleotides were synthesized, all targeted to the 5' cap region of
the eIF4E mRNA, i.e, the extreme 5' end of the mRNA adjacent to the 5'
cap. These are shown in Table 23. All cytosines are 5-methylcytosines.
While not wishing to be bound by theory, fully 2'-MOE oligonucleotides
are not believed to be substrates for RNAse H and thus are believed to
interfere with protein translation via an occupancy-only or steric
hindrance mechanism rather than via degradation of the mRNA target.

"Target site" refers to the position on the eIF4E mRNA (SEQ ID NO: 4 or
11 as indicated).

[0439] A series of PNA oligomers was also synthesized which are targeted
to the same sites as the oligonucleotides in Table 23. These are shown in
Table 24. Each has a lysine on the 3' end of the oligomer. As with the
fully modified 2' MOE compounds, PNA oligomers are not believed to be
substrates for RNAse H.

[0440] The duplexed oligomeric RNA (dsRNA) compounds shown in Table 14
below were prepared as described in previous examples and evaluated in
HeLa cells (American Type Culture Collection, Manassas Va.). Cells were
plated in 96-well plates at a density of 5000 cells/well and grown in
DMEM with high glucose, 10% FBS, 1% penicillin/streptomycin. Wells were
washed once with 200 μL OPTI-MEM-1® reduced-serum medium (Gibco
BRL) and then treated with 130 μL of OPTI-MEM-1® containing the
desired dsRNA at a concentration of 0.2, 2 and 20 nM plus 2.5 μl/ml
LIPOFECTIN® (Gibco BRL) per strand of oligomeric compound. Treatments
were done in duplicate. After 4 or 5 hours of treatment, the medium was
replaced with fresh medium. Cells were harvested 16 or 18 hours after
dsRNA treatment, at which time RNA was isolated and target reduction
measured by RT-PCR as described in previous examples.

[0441] The results are shown in Table 25. The siRNA constructs shown
consist of one antisense strand and one sense strand. The antisense
strand (AS) is shown first in Table 25 below, followed by the sense
strand (S) in the next row. Unless otherwise indicated, all
double-stranded constructs are unmodified RNA, i.e., ribose sugars with
phosphate (P═O) backbones and 5'-terminal hydroxyl group. It is
understood in the art that, for RNA sequences, U (uracil) generally
replaces T (thymine) which is normally found in DNA or DNA-like
sequences. 2'-O-methyl nucleosides are shown in bold. LNA nucleosides are
in italics.

[0454] siRNAs from Table 22 were tested for ability to reduce eIF4E RNA
levels in HeLa cells. These compounds were designed to human eIF4E mRNA
(Genbank accession no. M15353.1, SEQ ID NO:4). Unless noted, antisense
strands are alternating 2'-O-methyl/ribose starting with 2'-O-methyl at
position 1 (i.e., odd-numbered positions are 2'-O-methyl and
even-numbered positions are ribose) and sense strands are alternating
ribose/2'-O-methyl starting with ribose at position 1 (i.e., odd-numbered
positions are ribose and even-numbered positions are 2'-O-methyl). The
backbone is phosphate (P═O) and the 5' terminus is 5'-OH, although it
will be understood that these and other siRNA constructs shown herein may
also be synthesized with a 5'-phosphate group. Note that the ISIS
351831--351832 construct has the same sequence as the 345847345849
construct but the former is alternating 2'-O-methyl/2'-fluoro (antisense
strand has 2'-β-methyl on odd numbered positions and 2'-fluoro on
evens; sense strand has 2'F on odd numbered positions and 2'-O-methyl on
evens).

[0455] The compounds shown in Table 26 were tested at low dose of 5 nM in
HeLa cells as in above examples. The results are shown in Table 26. The
siRNA constructs shown consist of one antisense strand and one sense
strand. The antisense strand (AS) is shown first in the table below,
followed by the sense strand in the next row. It is understood in the art
that, for RNA sequences, U (uracil) generally replaces T (thymine) which
is normally found in DNA or DNA-like sequences. Results are shown as
percent reduction of eIF4E RNA ("% inhib") in Table 26. "Target site"
refers to the 5'-most position of the target region on the M15353.1 human
eIF4E sequence (SEQ ID NO: 4) to which the oligonucleotide is targeted.

[0456] A series of single-stranded RNA antisense oligonucleotides targeted
to human eIF4E (SEQ ID NO: 4) were synthesized. All are RNA (ribose
sugars) with phosphorothioate backbone linkages throughout and a 5'
phosphate cap. The human umbilical vein endothilial cell line HuVEC is
obtained from the American Type Culture Collection (Manassas, Va.). HuVEC
cells are routinely cultured in EBM (Clonetics Corporation Walkersville,
Md.) supplemented with SingleQuots supplements (Clonetics Corporation,
Walkersville, Md.). Cells are routinely passaged by trypsinization and
dilution when they reach 90% confluence and are maintained for up to 15
passages. Cells are seeded into 96-well plates (Falcon-Primaria #3872) at
a density of 10000 cells/well for treatment with RNA oligonucleotides (30
nM oligonucleotide concentration). Sequences and results of treatment
(reduction of eIF4E RNA levels) are shown in Table 27. It is understood
in the art that, for RNA sequences, U (uracil) generally replaces T
(thymine) which is normally found in DNA or DNA-like sequences. As in
above examples, "Target site" refers to the 5'-most position of the
target region on the M15353.1 human eIF4E sequence (SEQ ID NO: 4) to
which the oligonucleotide is targeted. "% inhib" refers to percent
reduction in eIF4E RNA (shown ±standard deviation).

[0457] As shown in the table above, all of the single-stranded antisense
RNA compounds were able to reduce human eIF4E RNA levels by at least 10%.
Compounds that reduced eIF4E RNA levels by at least 20%, at least 30%, at
least 40% or at least 50% are especially suitable for use as inhibitors
of eIF4E expression.

[0458] ISIS 347402 (SEQ ID NO: 228) gave the greatest reduction in eIF4E
expression in this experiment. A dose-response analysis of this
single-stranded antisense RNA compound was done in HeLa cells using
antisense RNA concentrations of 1 nM, 10 nM and 100 nM. ISIS 347402 gave
a dose-dependent inhibition of eIF4E expression, with 41% reduction of
eIF4E expression at 10 nM and 67% reduction at 100 nM (no effect was
observed at 1 nM dose in this experiment).

[0459] Double-stranded RNA compounds were prepared as in previous
examples. The antisense strands of the duplexes are identical in sequence
to the single-stranded antisense RNA compounds used in the previous
example, but were made with a phosphodiester (P═O) backbone. The
sense strand is fully complementary to the antisense strand (thus forming
a blunt ended 20mer duplex) and also has a P═O backbone. Both strands
are unmodified RNA. The siRNA duplexes were used at a concentration of 25
nM to treat HeLa cells as described in previous siRNA examples. Effect of
treatment on eIF4E RNA levels in HeLa cells is as shown in Table 28. "%
inhib" refers to percent reduction in eIF4E RNA (shown ±standard
deviation). Only the sequence of the antisense strand is shown in Table
28. It is understood in the art that, for RNA sequences, U (uracil)
generally replaces T (thymine) which is normally found in DNA or DNA-like
sequences.

[0461] Thus both single and double-stranded antisense RNA compounds are
able to cause inhibition of eIF4E RNA levels. Compounds which are active
in both single- and double-stranded versions (i.e, the active antisense
strand with or without a complementary sense strand) are believed to be
particularly useful.

[0462] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from the
foregoing description. Such modifications are also intended to fall
within the scope of the appended claims. Each reference (including, but
not limited to, journal articles, U.S. and non-U.S. patents, patent
application publications, international patent application publications,
gene bank accession numbers, and the like) cited in the present
application is incorporated herein by reference in its entirety. U.S.
provisional application Ser. No. 60/504,110 filed Sep. 18, 2004 and U.S.
provisional application Ser. No. 60/576,534 filed Jun. 3, 2004, are each
incorporated herein by reference in its entirety.